Splicing modulator antibody-drug conjugates and methods of use

ABSTRACT

Linker-drug compounds and antibody-drug conjugates that bind to human oncology targets are disclosed. The linker-drug compounds and antibody-drug conjugates comprise a splicing modulator drug moiety. The disclosure further relates to methods and compositions for use in the treatment of neoplastic disorders by administering the antibody-drug conjugates provided herein. In an embodiment, the splicing modulator comprises a pladienolide or a pladienolide derivative.

The present disclosure claims the benefit of priority to U.S.Provisional Patent Application No. 62/679,672, filed Jun. 1, 2018; U.S.Provisional Patent Application No. 62/679,631, filed Jun. 1, 2018; andU.S. Provisional Patent Application No. 62/779,324, filed Dec. 13, 2018.All of the aforementioned applications are incorporated herein byreference in their entirety.

The present disclosure relates to antibody-drug conjugates (ADCs)comprising a splicing modulator and an antibody or antigen bindingfragment thereof that binds a human oncology antigen target. Thedisclosure further relates to methods and compositions useful in thetreatment or diagnosis of cancers that express a target antigen and/orare amenable to treatment by disruption of RNA splicing, as well asmethods of making those compositions.

The majority of protein-coding genes in the human genome are composed ofmultiple exons (coding regions) that are separated by introns(non-coding regions). Gene expression results in a single precursormessenger RNA (pre-mRNA). The intron sequences are subsequently removedfrom the pre-mRNA by a process called splicing, which results in themature messenger RNA (mRNA). By including different combinations ofexons, alternative splicing gives rise to mRNAs encoding distinctprotein isoforms.

RNA splicing is catalyzed by the spliceosome, a dynamic multiprotein-RNAcomplex composed of five small nuclear RNAs (snRNAs U1, U2, U4, U5, andU6) and associated proteins. The spliceosome assembles on pre-mRNAs toestablish a dynamic cascade of multiple RNA and protein interactionsthat catalyze excision of the introns and ligation of exons (Matera andWang (2014) Nat Rev Mol Cell Biol. 15(2):108-21). Accumulating evidencehas linked human diseases to dysregulation in RNA splicing that impactmany genes (Scotti and Swanson (2016) Nat Rev Genet. 17(1):19-32).

The spliceosome is an important target in cancer biology. Severalstudies have now documented significant alterations in the splicingprofile of cancer cells, as well as in the splicing factors themselves(Agrawal et al. (2018) Curr Opin Genet Dev. 48:67-74). Alternativesplicing can lead to differential exon inclusion/exclusion, intronretention, or usage of cryptic splice sites (Seiler et al. (2018) CellRep. 23(1):282-296). Altogether, these events account for functionalchanges that may contribute to tumorigenesis or resistance to therapy(Siegfried and Karni (2018) Curr Opin Genet Dev. 48:16-21).

Certain natural products can bind the SF3b spliceosome complex. Thesesmall molecules modulate splicing by promoting intron retention and/orexon skipping (Teng et al. (2017) Nat Commun. 8:15522). A significantportion of the resulting transcripts contain premature stop codonstriggering nonsense mediated mRNA decay (NMD). Furthermore, becausecanonical splicing is impaired, canonical transcripts are considerablyreduced, which can negatively impact cell function and viability. Forthis reason, splicing modulators have become a promising class of drugsfor the treatment of cancer (Puthenveetil et al. (2016) BioconjugateChem. 27:1880-8).

The proto-oncogene human epidermal growth factor receptor 2 (HER2)encodes a transmembrane tyrosine kinase receptor that belongs to thehuman epidermal growth factor receptor (EGFR) family (King et al. (1985)Science 229:974-6). Overexpression of HER2 enables constitutiveactivation of growth factor signaling pathways, such as thePI3K-AKT-mTOR pathway, and thereby serves as an oncogenic driver inseveral types of cancers, including approximately 20% of invasive breastcarcinomas (Slamon et al. (1989) Science 244:707-12; Gajria andChandarlapaty (2011) Expert Rev Anticancer Ther. 11:263-75). Given thatHER2 amplification mediates the transformed phenotype, and because HER2expression is largely restricted to malignant cells, HER2 is a promisingantigen for targeting certain cancers and/or delivering novel cancertreatments (Parakh et al. (2017) Cancer Treat Rev. 59:1-21). Additionalantigens for targeted delivery of cancer therapies include, but are notlimited to, CD138 (also referred to as syndecan-1) and ephrin type-Areceptor 2 (EPHA2).

CD138 is a cell surface heparan sulfate proteoglycan that is essentialfor maintaining cell morphology and interaction with the surroundingmicroenvironment (Akl et al. (2015) Oncotarget 6(30):28693-715; Szatmáriet al. (2015) Dis Markers 2015:796052). In general, the loss of CD138expression in carcinoma cells reduces cell adhesion to the extracellularmatrix and enhances cell motility and invasion (Teng et al. (2012)Matrix Biol. 31:3-16). Increased stromal CD138 expression also altersfibronectin production and extracellular matrix organization (Yang etal. (2011) Am J Pathol. 178:325-35). Additionally, increased expressionof CD138 in stromal fibroblasts is associated with angiogenesis andcancer progression (Maeda et al. (2006) Oncogene 25:1408-12). CD138expression increases during B cell development and its presence is ahallmark of plasma cells (Ribatti (2017) Immunol Lett. 188:64-7). CD138expression is maintained in multiple myeloma, a malignancy of plasmacells. CD138 is therefore an attractive antigen for the targetedtreatment of several cancers and other hematological malignancies(Sherbenou et al. (2015) Blood Rev. 29(2):81-91; VVijdenes et al. (1996)Br J Haematol. 94(2):318-23).

EPHA2 is a transmembrane glycoprotein that is abundantly overexpressedin several malignant cancer-derived cell lines and in advanced forms ofcancer (Wykosky and Debinski (2008) Mol Cancer Ref. 6(12):1795-1806).For instance, EPHA2 is strongly overexpressed in approximately 61% ofGBM patient tumors (Wykosky et al. (2008) Clin Cancer Res. 14:199-208),76% of ovarian cancers (Thaker et al. (2004) Clin Cancer Res.10:5145-50), and 85% of prostate adenocarcinomas (Zeng et al. (2003) AmJ Pathol. 163:2271-6). The EPHA2 protein is highly overexpressed withregard to percentage of patient tumors and percentage of cells within atumor, and is a plasma membrane-localized receptor that can internalizeon ligand binding (Walker-Daniels et al. (2002) Mol Cancer Res.1:79-87). Moreover, expression of EPHA2 is associated with poorprognosis, increased metastasis, and decreased survival. Thus, due toits expression pattern, localization, and functional importance in theoutcome of cancer patients, EPHA2 is another attractive antigen for thetargeted delivery of novel anti-cancer therapies.

In various embodiments, the present disclosure provides, in part, novelcompounds with biological activity against neoplastic cells. Thecompounds may slow, inhibit, and/or reverse tumor growth in mammals, andmay be useful for treating human cancer patients. In variousembodiments, the disclosure provides novel antibody-drug conjugatesemploying the novel compounds or other functional splice inhibitormolecules.

The present disclosure more specifically relates, in variousembodiments, to antibody-drug conjugate (ADC) compounds that are capableof binding and killing neoplastic cells. In various embodiments, the ADCcompounds disclosed herein comprise a linker that attaches a splicingmodulator to a full-length antibody or an antigen binding fragment. Invarious embodiments, the ADC compounds are also capable of internalizinginto a target cell after binding.

In various embodiments, ADC compounds may be represented by Formula (I):

Ab-(L-D)_(p)  (I)

wherein Ab is an antibody or an antigen binding fragment thereof whichtargets a neoplastic cell or another oncology-related target;D is a splicing modulator;L is a linker which covalently attaches Ab to D; andp is an integer from 1 to 15.In various embodiments, ADC compounds may be represented by Formula (I):

Ab-(L-D)_(p)  (I)

wherein Ab is an antibody or an antigen binding fragment thereof whichtargets a neoplastic cell;D is a splicing modulator of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is chosen from absent, hydrogen, C₁-C₆ alkyl groups, C₁-C₆alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acidgroups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzylgroups, C₃-C₈ heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl) groups, and—CD₃;

R³ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxygroups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups,C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, and —O—C(═O)—(C₁-C₆ alkyl) groups; and

R⁴, R⁵, and R⁸ are each independently chosen from hydrogen, hydroxylgroups, —O—(C₁-C₆ alkyl) groups, —O—C(═O)—(C₁-C₆ alkyl) groups, andC₁-C₆ alkyl groups;

R⁶ and R⁷ are each independently chosen from hydrogen, —O—R¹⁷,—O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₆ alkyl groups, and —NR¹⁵R¹⁶;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷;

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups; and

Z is chosen from

wherein R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, R¹⁶, and R¹⁷ are eachindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups,

—O—(C₁-C₆ alkyl) groups, —NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆alkylhydroxy groups, C₁-C₆ alkylalkoxy groups, benzyl groups, and C₃-C₈heterocyclyl groups,wherein at least one of R⁶ and R⁷ is hydrogen;and wherein L is a linker which covalently attaches Ab to D; andp is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment is capableof internalization into a target cell. In some embodiments, the linkercovalently attaches to the splicing modulator of Formula (II) (“L-D”),and L-D has a structure of Formula (II-A):

or a pharmaceutically acceptable salt thereof,

wherein Z′ is chosen from

and

wherein all other variables are as defined for Formula (II).

In various other embodiments, ADC compounds may be represented by ofFormula (I):

Ab-(L-D)_(p)  (I)

wherein Ab is an antibody or an antigen binding fragment thereof whichtargets a neoplastic cell;D is a splicing modulator of Formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxygroups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups,C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl) groups, and —CD₃;

R³ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxygroups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups,C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, and —O—C(═O)—(C₁-C₆ alkyl) groups; and

R⁴, R⁵, and R⁸ are each independently chosen from hydrogen, hydroxylgroups, —O—(C₁-C₆ alkyl) groups, —O—C(═O)—(C₁-C₆ alkyl) groups, andC₁-C₆ alkyl groups;

R⁶ and R⁷ are each independently chosen from hydrogen, —O—R¹⁷,—O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₆ alkyl groups, and —NR¹⁵R¹⁶;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷; and

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, R¹⁶, and R¹⁷ are eachindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups,—NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆alkylalkoxy groups, benzyl groups, and C₃-C₈ heterocyclyl groups,

wherein at least one of R⁶ and R⁷ is hydrogen;

and wherein L is a linker which covalently attaches Ab to D; andp is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment is capableof internalization into a target cell. In some embodiments, the linkercovalently attaches to the splicing modulator (“L-D”), and L-D has astructure of Formula (IV-A):

or a pharmaceutically acceptable salt thereof.

In various other embodiments, ADC compounds may be represented byFormula (I):

Ab-(L-D)_(p)  (I)

wherein Ab is an antibody or an antigen binding fragment thereof whichtargets a neoplastic cell;D is a splicing modulator of Formula (VI):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R⁹ are each independently chosen from hydrogen, C₁-C₆ alkylgroups, C₁-C₆ alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆alkylcarboxylic acid groups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkylgroups, benzyl groups, C₃-C₈ heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl)groups, and —CD₃;

R³ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxygroups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups,C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, and —O—C(═O)—(C₁-C₆ alkyl) groups;

R⁴, R⁵, and R⁸ are each independently chosen from hydrogen, hydroxylgroups, —O—(C₁-C₆ alkyl) groups, —O—C(═O)—(C₁-C₆ alkyl) groups, andC₁-C₆ alkyl groups;

R⁶ and R⁷ are each independently chosen from hydrogen, —O—R¹⁷,—O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₆ alkyl groups, —NR¹⁵R¹⁶, and alinker;

R¹⁰ is chosen from hydrogen, C₁-C₆ alkyl groups, —C(═O)—(C₁-C₆ alkyl)groups, and —CD₃;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷;

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups; and

a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

wherein R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹⁵, R¹⁶, and R¹⁷ are eachindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups,—NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆alkylalkoxy groups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein at least one of R⁶ and R⁷ is hydrogen; and

wherein R¹ and R⁹ cannot both be absent;

and wherein L is a linker which covalently attaches Ab to D; andp is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment is capableof internalization into a target cell. In some embodiments, the linkercovalently attaches to the splicing modulator (“L-D”), and L-D has astructure of Formula (VI-A):

or a pharmaceutically acceptable salt thereof.

In various other embodiments, ADC compounds may be represented byFormula (I):

Ab-(L-D)_(p)  (I)

wherein Ab is an antibody or an antigen binding fragment thereof whichtargets a neoplastic cell;D is a splicing modulator of Formula (VIII):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is chosen from absent, hydrogen, C₁-C₆ alkyl groups, C₁-C₆alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acidgroups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzylgroups, C₃-C₈ heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl) groups, and—CD₃;

R³ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxygroups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups,C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, and —O—C(═O)—(C₁-C₆ alkyl) groups;

R⁴ is chosen from hydrogen, hydroxyl groups, —O—(C₁-C₆ alkyl) groups,—O—C(═O)—(C₁-C₆ alkyl) groups, and C₁-C₆ alkyl groups; and

R¹⁰ is chosen from 3 to 10 membered carbocycles and 3 to 10 memberedheterocycles, each of which is substituted with 0 to 3 R^(a), whereineach R^(a) is independently chosen from halogens, C₁-C₆ alkyl groups,—O—(C₁-C₆)alkyl groups, C₁-C₆ alkylalkoxy groups, C₁-C₆ alkylhydroxygroups, —S(═O)_(w)-(4 to 7 membered heterocycles), 4 to 7 memberedcarbocycles, and 4 to 7 membered heterocycles;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷; and

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein R¹, R³, R⁴, R¹⁰, R¹⁵, R¹⁶, and R¹⁷ are each independentlysubstituted with 0 to 3 groups independently chosen from halogens,hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups, —NR¹⁵R¹⁶,C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆ alkylalkoxygroups, benzyl groups, and C₃-C₈ heterocyclyl groups; and wherein eachR^(a) is independently substituted with 0 to 3 groups independentlychosen from halogens, hydroxyl groups, —NR¹⁵R¹⁶, C₁-C₆ alkyl groups,—(C═O)—(C₁-C₆ alkyl) groups, —(C═O)—(C₁-C₆ alkyl)-(C₃-C₁₀ heterocyclylgroups), —S(═O)_(w)—(C₃-C₈ heterocyclyl) groups, and C₁-C₆alkylcarboxylic acid groups, each of which is substituted with 0, 1, or2 groups independently chosen from halogens, hydroxyl groups, —NR¹⁵R¹⁶,and C₁-C₃ alkyl groups; and

w is 0, 1, or 2;

and wherein L is a linker which covalently attaches Ab to D; andp is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment is capableof internalization into a target cell. In some embodiments, the linkercovalently attaches to the splicing modulator (“L-D”), and L-D has astructure of Formula (VIII-A):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the splicing modulator comprises a modulator of theSF3b complex.

In some embodiments, the splicing modulator comprises a pladienolide ora pladienolide derivative. In some embodiments, the splicing modulatorcomprises pladienolide D or a pladienolide D derivative. In someembodiments, the pladienolide D or derivative comprises D2, D1, D4, D8,D10, D11 (E7107), D20, D21, D22, D12, or D25. In some embodiments, thepladienolide D or derivative comprises D2. In some embodiments, thepladienolide D or derivative comprises D1. In some embodiments, thepladienolide D or derivative comprises D4. In some embodiments, thepladienolide D or derivative comprises D12.

In some embodiments, the pladienolide D or derivative is a zwitterionicpladienolide D or derivative. In some embodiments, the zwitterionicpladienolide D or derivative comprises D22 or D25.

In some other embodiments, the splicing modulator comprises pladienolideB or a pladienolide B derivative. In some embodiments, the pladienolideB or derivative comprises D9, D18, D19, or D13.

In some embodiments, the splicing modulator comprises an arylpladienolide. In some embodiments, the aryl pladienolide comprises D15,D14, D16, D17, D26, or D33. In some embodiments, the aryl pladienolidecomprises D15. In some embodiments, the aryl pladienolide is azwitterionic aryl pladienolide. In some embodiments, the zwitterionicaryl pladienolide comprises D33.

In some embodiments, the splicing modulator comprises D1:

In some embodiments, the splicing modulator comprises D2:

In some embodiments, the splicing modulator comprises D3:

In some embodiments, the splicing modulator comprises D4:

In some embodiments, the splicing modulator comprises D4′:

In some embodiments, the splicing modulator comprises D5:

In some embodiments, the splicing modulator comprises D6:

In some embodiments, the splicing modulator comprises D7:

In some embodiments, the splicing modulator comprises D8:

In some embodiments, the splicing modulator comprises D9:

In some embodiments, the splicing modulator comprises D10:

In some embodiments, the splicing modulator comprises D11:

In some embodiments, the splicing modulator comprises D12:

In some embodiments, the splicing modulator comprises D13:

In some embodiments, the splicing modulator comprises D14:

In some embodiments, the splicing modulator comprises D15:

In some embodiments, the splicing modulator comprises D16:

In some embodiments, the splicing modulator comprises D17:

In some embodiments, the splicing modulator comprises D18:

In some embodiments, the splicing modulator comprises D19:

In some embodiments, the splicing modulator comprises D20:

In some embodiments, the splicing modulator comprises D21:

In some embodiments, the splicing modulator comprises D22:

In some embodiments, the splicing modulator comprises D23:

In some embodiments, the splicing modulator comprises D24:

In some embodiments, the splicing modulator comprises D25:

In some embodiments, the splicing modulator comprises D26:

In some embodiments, the splicing modulator comprises D27:

In some embodiments, the splicing modulator comprises D28:

In some embodiments, the splicing modulator comprises D29:

In some embodiments, the splicing modulator comprises D30:

In some embodiments, the splicing modulator comprises D31:

In some embodiments, the splicing modulator comprises D32:

In some embodiments, the splicing modulator comprises D33:

In some embodiments, the splicing modulator comprises D34:

In some embodiments, the splicing modulator comprises D35:

In some embodiments, the splicing modulator comprises one of the drugmoieties listed in Table 7. In some embodiments, the splicing modulatorcomprises D1, D2, D3, D4, D4′, D5, D6, D7, D8, D9, D10, D11, D12, D13,D14, D15, D16, D17, D18, D19, D20, D21, D22, D23, D24, D25, D26, D27,D28, D29, D30, D31, D32, D33, D34, and/or D35.

In some embodiments, a splicing modulator is disclosed, as well as itsuse as a therapeutic alone or as part of an ADC. In some embodiments,the splicing modulator comprises D4, D4′, D12, D15, D8, D9, D10, D13,D18, D19, D20, D21, D22, D25, or D33.

In some embodiments, the splicing modulator comprises D4 and the linkercomprises MC-Val-Cit-pABC. In some embodiments, the splicing modulatorcomprises D4 and the linker comprises MC-3-glucuronide. In someembodiments, the splicing modulator comprises D12 and the linkercomprises MC-Val-Cit-pABC. In some embodiments, the splicing modulatorcomprises D12 and the linker comprises MC-β-glucuronide. In someembodiments, the splicing modulator comprises D15 and the linkercomprises MC-Val-Ala-pAB.

In various embodiments, the linker used in an ADC disclosed herein isstable outside a cell, such that the ADC remains intact when present inextracellular conditions, but is capable of being cleaved uponinternalization into a cell, e.g., a tumor or cancer cell. In someembodiments, the splicing modulator is cleaved from the antibody orantigen binding fragment when the ADC enters a cell that expresses anantigen targeted by the antibody or antigen binding fragment of the ADC.In some embodiments, the linker is a cleavable linker.

In some embodiments, the linker comprises a cleavable peptide moiety. Insome embodiments, the cleavable peptide moiety is cleavable by anenzyme. In some embodiments, the cleavable peptide moiety or linkercomprises an amino acid unit. In some embodiments, the amino acid unitcomprises valine-citrulline (“Val-Cit” or “VC”). In some otherembodiments, the amino acid unit comprises valine-alanine (“Val-Ala” or“VA”). In some other embodiments, the amino acid unit comprises glutamicacid-valine-citrulline (“Glu-Val-Cit” or “EVC”). In some otherembodiments, the amino acid unit comprises alanine-alanine-asparagine(“Ala-Ala-Asn” or “AAN”).

In some embodiments, the linker comprises a cleavable glucuronidemoiety. In some embodiments, the cleavable glucuronide moiety iscleavable by an enzyme. In some embodiments, the cleavable glucuronidemoiety is cleavable by a glucuronidase. In some embodiments, thecleavable glucuronide moiety is cleavable by β-glucuronidase.

In some embodiments, the linker comprises at least one spacer unit. Insome embodiments, the spacer unit or linker comprises a polyethyleneglycol (PEG) moiety. In some embodiments, the PEG moiety comprises-(PEG)_(m)- and m is an integer from 1 to 10. In some embodiments, m is2. In some other embodiments, the spacer unit or linker comprises analkyl moiety. In some embodiments, the alkyl moiety comprises—(CH₂)_(n)— and n is an integer from 1 to 10. In some embodiments, n is2. In some embodiments, n is 5. In some embodiments, n is 6.

In some embodiments, the spacer unit attaches to the antibody or antigenbinding fragment via a maleimide (Mal) moiety (“Mal-spacer unit”). Insome embodiments, the Mal-spacer unit is reactive with a cysteineresidue on the antibody or antigen binding fragment. In someembodiments, the Mal-spacer unit is joined to the antibody or antigenbinding fragment via a cysteine residue on the antibody or antigenbinding fragment.

In some embodiments, the linker comprises the Mal-spacer unit and acleavable peptide moiety. In some embodiments, the cleavable peptidemoiety comprises an amino acid unit. In some embodiments, the cleavablepeptide moiety or amino acid unit comprises Val-Cit. In someembodiments, the cleavable peptide moiety or amino acid unit comprisesVal-Ala. In some embodiments, the cleavable peptide moiety or amino acidunit comprises Glu-Val-Cit. In some embodiments, the cleavable peptidemoiety or amino acid unit comprises Ala-Ala-Asn. In some embodiments,the Mal-spacer unit comprises an alkyl moiety. In some embodiments, theMal-spacer unit comprises a PEG moiety. In some embodiments, theMal-spacer unit comprises a maleimidocaproyl (MC).

In some embodiments, the Mal-spacer unit attaches the antibody orantigen binding fragment to the cleavable moiety in the linker. In someembodiments, the cleavable moiety in the linker comprises a cleavablepeptide moiety. In some embodiments, the cleavable peptide moietycomprises an amino acid unit. In some embodiments, the cleavable peptidemoiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, orAla-Ala-Asn. In some embodiments, the linker comprises MC-Val-Cit. Insome embodiments, the linker comprises MC-Val-Ala. In some embodiments,the linker comprises MC-Glu-Val-Cit. In some embodiments, the linkercomprises MC-Ala-Ala-Asn. In some embodiments, the Mal-spacer unitcomprises an alkyl moiety. In some embodiments, the Mal-spacer unitcomprises a PEG moiety. In some embodiments, the Mal-spacer unitcomprises a maleimidocaproyl (MC).

In some embodiments, the cleavable moiety in the linker is directlyjoined to the splicing modulator, or a spacer unit attaches thecleavable moiety in the linker to the splicing modulator. In someembodiments, cleavage of the conjugate releases the splicing modulatorfrom the antibody or antigen binding fragment and linker. In someembodiments, the spacer unit attaching the cleavable moiety in thelinker to the splicing modulator is self-immolative.

In some embodiments, the spacer unit attaching the cleavable moiety inthe linker to the splicing modulator comprises ap-aminobenzyloxycarbonyl (pABC). In some embodiments, the pABC attachesthe cleavable moiety in the linker to the splicing modulator. In someembodiments, the cleavable moiety in the linker comprises a cleavablepeptide moiety. In some embodiments, the cleavable peptide moietycomprises an amino acid unit. In some embodiments, the cleavable peptidemoiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, orAla-Ala-Asn. In some embodiments, the linker comprises Val-Cit-pABC. Insome other embodiments, the linker comprises Val-Ala-pABC. In someembodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments,the linker comprises Ala-Ala-Asn-pABC.

In some embodiments, the spacer unit attaching the cleavable moiety inthe linker to the splicing modulator comprises a p-aminobenzyl (pAB). Insome embodiments, the pAB attaches the cleavable moiety in the linker tothe splicing modulator. In some embodiments, the cleavable moiety in thelinker comprises a cleavable peptide moiety. In some embodiments, thecleavable peptide moiety comprises an amino acid unit. In someembodiments, the cleavable peptide moiety or amino acid unit comprisesVal-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, thelinker comprises Val-Cit-pAB. In some other embodiments, the linkercomprises Val-Ala-pAB. In some other embodiments, the linker comprisesGlu-Val-Cit-pAB. In some other embodiments, the linker comprisesAla-Ala-Asn-pAB.

In various embodiments, the linker is a non-cleavable linker. In someembodiments, the splicing modulator of the ADC is released bydegradation of the antibody or antigen binding fragment. In someembodiments, the linker remains covalently associated with at least oneamino acid of the antibody and drug upon internalization by anddegradation within the target cell.

In some embodiments, the linker is a non-cleavable linker comprising atleast one spacer unit. In some embodiments, the spacer unit or linkercomprises a polyethylene glycol (PEG) moiety. In some embodiments, thePEG moiety comprises -(PEG)_(m)- and m is an integer from 1 to 10. Insome embodiments, m is 2. In some other embodiments, the spacer unit orlinker comprises an alkyl moiety. In some embodiments, the alkyl moietycomprises —(CH₂)_(n)— or —(CH₂)_(n)—O—(CH₂), and n is an integer from 1to 10. In some embodiments, n is 2. In some embodiments, n is 5. In someembodiments, n is 6.

In some embodiments, the spacer unit in a non-cleavable linker attachesto the antibody or antigen binding fragment via a maleimide (Mal) moiety(“Mal-spacer unit”). In some embodiments, the Mal-spacer unit isreactive with a cysteine residue on the antibody or antigen bindingfragment. In some embodiments, the Mal-spacer unit is joined to theantibody or antigen binding fragment via a cysteine residue on theantibody or antigen binding fragment. In some embodiments, theMal-spacer unit comprises an alkyl moiety. In some embodiments, theMal-spacer unit comprises a PEG moiety. In some embodiments, the linkeror Mal-spacer unit comprises a maleimidocaproyl (MC). In someembodiments, the linker or Mal-spacer unit comprises a maleimidocaproyl(MC) and at least one additional spacer unit. In some embodiments, thelinker or Mal-spacer unit comprises MC-(PEG)₂. In some embodiments, thelinker or Mal-spacer unit comprises MC-(PEG)₂ and at least oneadditional spacer unit. In some embodiments, the linker or Mal-spacerunit comprises Mal-Hex. In some embodiments, the linker or Mal-spacerunit comprises Mal-Hex and at least one additional spacer unit. In someembodiments, the linker or Mal-spacer unit comprises Mal-Et. In someembodiments, the linker or Mal-spacer unit comprises Mal-Et and at leastone additional spacer unit. In some embodiments, the linker orMal-spacer unit comprises Mal-Et-O-Et. In some embodiments, the linkeror Mal-spacer unit comprises Mal-Et-O-Et and at least one additionalspacer unit. In some embodiments, the Mal-spacer unit attaches theantibody or antigen binding fragment to the splicing modulator.

In various embodiments, ADC compounds may be represented by Formula (I):

Ab-(L-D)_(p)  (I)

wherein Ab is an antibody or an antigen binding fragment thereof whichtargets a neoplastic cell or another oncology-related target such as acancer antigen (e.g., any of the antibody or binding domain sequencesdisclosed herein); D is any small molecule suitable for treating acancer (e.g., a splicing modulator, e.g., any of the splicing modulatorsdisclosed herein); L is a linker which covalently attaches Ab to D(e.g., any of the linkers disclosed herein); and p is an integer from 1to 15.

In some embodiments, Ab is selected from any of the antibody or bindingdomain sequences disclosed herein. In some embodiments, Ab is anantibody or binding domain sequence which targets HER2 and/or aHER2-expressing neoplastic cell. In some embodiments, Ab is an antibodyor binding domain sequence which targets CD138 and/or a CD138-expressingneoplastic cell. In some embodiments, Ab is an antibody or bindingdomain sequence which targets EPHA2 and/or an EPHA2-expressingneoplastic cell. In some embodiments, Ab is an antibody or bindingdomain sequence which targets MSLN and/or a MSLN-expressing neoplasticcell. In some embodiments, Ab is an antibody or binding domain sequencewhich targets FOLH1 and/or a FOLH1-expressing neoplastic cell. In someembodiments, Ab is an antibody or binding domain sequence which targetsCDH6 and/or a CDH6-expressing neoplastic cell. In some embodiments, Abis an antibody or binding domain sequence which targets CEACAM5 and/or aCEACAM5-expressing neoplastic cell. In some embodiments, Ab is anantibody or binding domain sequence which targets CFC1B and/or aCFC1B-expressing neoplastic cell. In some embodiments, Ab is an antibodyor binding domain sequence which targets ENPP3 and/or anENPP3-expressing neoplastic cell. In some embodiments, Ab is an antibodyor binding domain sequence which targets FOLR1 and/or a FOLR1-expressingneoplastic cell. In some embodiments, Ab is an antibody or bindingdomain sequence which targets HAVCR1 and/or a HAVCR1-expressingneoplastic cell. In some embodiments, Ab is an antibody or bindingdomain sequence which targets KIT and/or a KIT-expressing neoplasticcell. In some embodiments, Ab is an antibody or binding domain sequencewhich targets MET and/or a MET-expressing neoplastic cell. In someembodiments, Ab is an antibody or binding domain sequence which targetsMUC16 and/or a MUC16-expressing neoplastic cell. In some embodiments, Abis an antibody or binding domain sequence which targets SLC39A6 and/or aSLC39A6-expressing neoplastic cell. In some embodiments, Ab is anantibody or binding domain sequence which targets SLC44A4 and/or aSLC44A4-expressing neoplastic cell. In some embodiments, Ab is anantibody or binding domain sequence which targets STEAP1 and/or aSTEAP1-expressing neoplastic cell. In some embodiments, Ab is anantibody or binding domain sequence which targets another cancerantigen.

In some embodiments, D is a splicing modulator. In some embodiments, Dis selected from any of the splicing modulators disclosed herein. Insome embodiments, D is a splicing modulator selected from D2, D1, D4,D8, D10, D11 (E7107), D20, D21, D22, D12, D25, D9, D18, D19, D13, D15,D14, D16, D17, D26, and D33, or any derivative thereof. In someembodiments, D is a splicing modulator selected from D4, D12, D15, D8,D9, D10, D13, D18, D19, D20, D21, D22, D25, and D33, or any derivativethereof. In some embodiments, D is a splicing modulator comprising D2 orany derivative thereof. In some embodiments, D is a splicing modulatorcomprising D1 or any derivative thereof.

In some embodiments, L is selected from any of the linkers disclosedherein, or any combination of linker components disclosed herein. Insome embodiments, L is a linker comprising MC-Val-Cit-pABC,Mal-(PEG)₂-CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB, Mal-Hex,Mal-Et, or Mal-Et-O-Et. In some embodiments, the linker may alsocomprise one or more additional spacer units. In some embodiments, L isan ADL1, ADL2, ADL5, ADL6, ADL7, ADL10, ADL12, ADL13, ADL14, ADL15,ADL21, ADL22, or ADL23 linker. In some embodiments, L is an ADL1, ADL2,ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker. Insome embodiments, L is an ADL12, ADL14, or ADL15 linker. In someembodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14,ADL21, ADL23, or ADL15 linker may also comprise one or more additionalspacer units. In some embodiments, L is an ADL1 linker and mayoptionally comprise one or more additional spacer units. In someembodiments, L is an ADL2 linker and may optionally comprise one or moreadditional spacer units. In some embodiments, L is an ADL5 linker andmay optionally comprise one or more additional spacer units. In someembodiments, L is an ADL6 linker and may optionally comprise one or moreadditional spacer units. In some embodiments, L is an ADL7 linker andmay optionally comprise one or more additional spacer units. In someembodiments, L is an ADL12 linker and may optionally comprise one ormore additional spacer units. In some embodiments, L is an ADL14 linkerand may optionally comprise one or more additional spacer units. In someembodiments, L is an ADL15 linker and may optionally comprise one ormore additional spacer units. In various embodiments of the ADCsdescribed herein, p is from 1 to 10. In various embodiments, p is from 2to 8. In various embodiments, p is from 4 to 8. In some embodiments, pis 4. In some embodiments, p is 8.

In some embodiments, L-D of Formula (I) is ADL1-D1. In some embodiments,L-D of Formula (I) is ADL6-D1. In some embodiments, L-D of Formula (I)is ADL5-D2. In some embodiments, L-D of Formula (I) is ADL1-D18. In someembodiments, L-D of Formula (I) is ADL5-D19. In some embodiments, L-D ofFormula (I) is ADL14-D1. In some embodiments, L-D of Formula (I) isADL12-D1. In some embodiments, L-D of Formula (I) is ADL15-D1. In someembodiments, L-D of Formula (I) is ADL12-D20. In some embodiments, L-Dof Formula (I) is ADL10-D1. In some embodiments, L-D of Formula (I) isADL12-D2. In some embodiments, L-D of Formula (I) is ADL15-D2. In someembodiments, L-D of Formula (I) is ADL12-D21. In some embodiments, L-Dof Formula (I) is ADL6-D9. In some embodiments, L-D of Formula (I) isADL1-D4. In some embodiments, L-D of Formula (I) is ADL1-D3. In someembodiments, L-D of Formula (I) is ADL1-D12. In some embodiments, L-D ofFormula (I) is ADL1-D7. In some embodiments, L-D of Formula (I) isADL1-D6. In some embodiments, L-D of Formula (I) is ADL1-D5. In someembodiments, L-D of Formula (I) is ADL22-D4. In some embodiments, L-D ofFormula (I) is ADL5-D10. In some embodiments, L-D of Formula (I) isADL5-D11. In some embodiments, L-D of Formula (I) is ADL1-D13. In someembodiments, L-D of Formula (I) is ADL1-D8. In some embodiments, L-D ofFormula (I) is ADL1-D22. In some embodiments, L-D of Formula (I) isADL5-D25. In some embodiments, L-D of Formula (I) is ADL12-D22. In someembodiments, L-D of Formula (I) is ADL5-D15. In some embodiments, L-D ofFormula (I) is ADL1-D14. In some embodiments, L-D of Formula (I) isADL5-D26. In some embodiments, L-D of Formula (I) is ADL1-D16. In someembodiments, L-D of Formula (I) is ADL5-D17. In some embodiments, L-D ofFormula (I) is ADL1-D33. In some embodiments, L-D of Formula (I) isADL1-D28. In some embodiments, L-D of Formula (I) is ADL1-D31. In someembodiments, L-D of Formula (I) is ADL1-D29. In some embodiments, L-D ofFormula (I) is ADL1-D35. In some embodiments, L-D of Formula (I) isADL5-D32. In some embodiments, L-D of Formula (I) is ADL5-D27. In someembodiments, L-D of Formula (I) is ADL12-D35. In some embodiments, L-Dof Formula (I) is ADL12-D28. In some embodiments, L-D of Formula (I) isADL1-D23. In some embodiments, L-D of Formula (I) is ADL1-D24.

In some embodiments, a pool of ADCs is provided whereby randomconjugation occurs, and the average p in the pool is between about 2 andabout 8. In some embodiments, a pool of ADCs is provided whereby randomconjugation occurs, and the average p in the pool is between about 4 andabout 8. In some embodiments, a pool of ADCs is provided whereby randomconjugation occurs, and the average p in the pool is about 4. In someembodiments, a pool of ADCs is provided whereby random conjugationoccurs, and the average p in the pool is about 8. Compositions (e.g.,pharmaceutical compositions) comprising multiple copies of any of thedescribed ADCs, wherein the average drug loading (average p) of the ADCsin the composition is from about 3.5 to about 5.5 (e.g., about 4), orfrom about 7 to about 9 (e.g., about 8) are provided herein.

In some embodiments, the antibody or antigen binding fragment (Ab) ofthe ADC targets a neoplastic cell derived from a hematologicalmalignancy or a solid tumor. In some embodiments, the antibody orantigen binding fragment targets a neoplastic cell derived from ahematological malignancy. In some embodiments, the hematologicalmalignancy is selected from a B-cell malignancy, a leukemia (e.g., acutemyeloid leukemia), a lymphoma, and a myeloma (e.g., multiple myeloma).In some embodiments, the hematological malignancy is selected from acutemyeloid leukemia and multiple myeloma. In some embodiments, the antibodyor antigen binding fragment targets a neoplastic cell derived from asolid tumor. In some embodiments, the solid tumor is selected frombreast cancer (e.g., HER2-positive breast cancer), gastric cancer (e.g.,gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer(e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serousendometrial carcinoma), salivary duct carcinoma, melanoma, colon cancer,cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer,and esophageal cancer. In some embodiments, the solid tumor is selectedfrom HER2-positive breast cancer, gastric adenocarcinoma, prostatecancer, and osteosarcoma.

In various embodiments, the antibody or antigen binding fragment (Ab) ofthe ADC is an anti-HER2 antibody or an antigen binding fragment thereof.In some embodiments, the antibody or antigen binding fragment binds toHER2 and targets HER2-expressing neoplastic cells (i.e., the ADC targetsHER2-expressing neoplastic cells). In some embodiments, the antibody orantigen binding fragment of the ADC is an internalizing anti-HER2antibody or internalizing antigen binding fragment thereof.

In some embodiments, the anti-HER2 antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:1(HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5(LCDR2), and SEQ ID NO:6 (LCDR3). In some embodiments, the anti-HER2antibody or antigen binding fragment is an internalizing antibody orinternalizing antigen binding fragment. In some embodiments, theanti-HER2 antibody or antigen binding fragment comprises human frameworksequences. In some embodiments, the anti-HER2 antibody or antigenbinding fragment comprises a heavy chain variable region comprising anamino acid sequence of SEQ ID NO:19, and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO:20. In some embodiments,the anti-HER2 antibody or antigen binding fragment comprises a human IgGheavy chain constant region. In some embodiments, the anti-HER2 antibodyor antigen binding fragment comprises a human IgG1 heavy chain constantregion. In some embodiments, the anti-HER2 antibody or antigen bindingfragment comprises a human Ig kappa or lambda light chain constantregion. In some embodiments, the anti-HER2 antibody or antigen bindingcompetes for binding and/or binds the same epitope as an antibodycomprising a heavy chain variable domain of SEQ ID NO:19 and a lightchain variable domain of SEQ ID NO:20.

In various embodiments, the antibody or antigen binding fragment (Ab) ofthe ADC is an anti-CD138 antibody or an antigen binding fragmentthereof. In some embodiments, the antibody or antigen binding fragmentbinds to CD138 and targets CD138-expressing neoplastic cells (i.e., theADC targets CD138-expressing neoplastic cells). In some embodiments, theantibody or antigen binding fragment of the ADC is an internalizinganti-CD138 antibody or internalizing antigen binding fragment thereof.

In some embodiments, the anti-CD138 antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:7(HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11(LCDR2), and SEQ ID NO:12 (LCDR3). In some embodiments, the anti-CD138antibody or antigen binding fragment is an internalizing antibody orinternalizing antigen binding fragment. In some embodiments, theanti-CD138 antibody or antigen binding fragment comprises humanframework sequences. In some embodiments, the anti-CD138 antibody orantigen binding fragment comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO:21, and a light chainvariable region comprising an amino acid sequence of SEQ ID NO:22. Insome embodiments, the anti-CD138 antibody or antigen binding fragmentcomprises a murine IgG2a heavy chain constant region. In someembodiments, the anti-CD138 antibody or antigen binding fragmentcomprises a murine Ig kappa light chain constant region. In someembodiments, the anti-CD138 antibody or antigen binding fragmentcomprises a human IgG heavy chain constant region. In some embodiments,the anti-CD138 antibody or antigen binding fragment comprises a humanIgG2a heavy chain constant region. In some embodiments, the anti-CD138antibody or antigen binding fragment comprises a human Ig kappa orlambda light chain constant region. In some embodiments, the anti-CD138antibody or antigen binding competes for binding and/or binds the sameepitope as an antibody comprising a heavy chain variable domain of SEQID NO:21 and a light chain variable domain of SEQ ID NO:22.

In various embodiments, the antibody or antigen binding fragment (Ab) ofthe ADC is an anti-EPHA2 antibody or an antigen binding fragmentthereof. In some embodiments, the antibody or antigen binding fragmentbinds to EPHA2 and targets EPHA2-expressing neoplastic cells (i.e., theADC targets EPHA2-expressing neoplastic cells). In some embodiments, theantibody or antigen binding fragment of the ADC is an internalizinganti-EPHA2 antibody or internalizing antigen binding fragment thereof.

In some embodiments, the anti-EPHA2 antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:13(HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17(LCDR2), and SEQ ID NO:18 (LCDR3). In some embodiments, the anti-EPHA2antibody or antigen binding fragment is an internalizing antibody orinternalizing antigen binding fragment. In some embodiments, theanti-EPHA2 antibody or antigen binding fragment comprises humanframework sequences. In some embodiments, the anti-EPHA2 antibody orantigen binding fragment comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO:23, and a light chainvariable region comprising an amino acid sequence of SEQ ID NO:24. Insome embodiments, the anti-EPHA2 antibody or antigen binding fragmentcomprises a human IgG heavy chain constant region. In some embodiments,the anti-EPHA2 antibody or antigen binding fragment comprises a humanIgG1 heavy chain constant region. In some embodiments, the anti-EPHA2antibody or antigen binding fragment comprises a human Ig kappa orlambda light chain constant region. In some embodiments, the anti-EPHA2antibody or antigen binding competes for binding and/or binds the sameepitope as an antibody comprising a heavy chain variable domain of SEQID NO:23 and a light chain variable domain of SEQ ID NO:24.

Also provided herein, in various embodiments, are compounds comprising alinker-drug defined by the generic formula: L-D, wherein L=a linkermoiety, and D=a drug moiety (e.g., a splicing modulator drug moiety). Invarious embodiments, the linker-drug (L-D) compounds disclosed hereinmay attach to an antibody or antigen binding fragment and/or aresuitable for use in the ADCs disclosed herein, e.g., in ADCs of Formula(I).

In various embodiments, the linker-drug (L-D) compounds disclosed hereincomprise a linker-drug structure according to Formula (III). In variousembodiments, the present disclosure provides a linker-drug (L-D)compound of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is chosen from absent, hydrogen, C₁-C₆ alkyl groups, C₁-C₆alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acidgroups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzylgroups, C₃-C₈ heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl) groups, and—CD₃;

R² is absent or a linker;

R³ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxygroups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups,C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, and —O—C(═O)—(C₁-C₆ alkyl) groups; and

R⁴, R⁵, and R⁸ are each independently chosen from hydrogen, hydroxylgroups, —O—(C₁-C₆ alkyl) groups, —O—C(═O)—(C₁-C₆ alkyl) groups, andC₁-C₆ alkyl groups;

R⁶ and R⁷ are each independently chosen from hydrogen, —O—R¹⁷,—O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₆ alkyl groups, —NR¹⁵R¹⁶, and alinker;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷;

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups; and

Z″ is chosen from

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, R¹⁶, and R¹⁷ are eachindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups,—NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆alkylalkoxy groups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein at least one of R⁶ and R⁷ is hydrogen; and

wherein if R² is a linker, then neither R⁶ or R⁷ is a linker, and if R⁶or R⁷ is a linker, then R² is absent.

In various other embodiments, the linker-drug (L-D) compounds disclosedherein comprise a linker-drug structure according to Formula (V). Invarious embodiments, the present disclosure provides a linker-drug (L-D)compound of Formula (V):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is chosen from absent, hydrogen, C₁-C₆ alkyl groups, C₁-C₆alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acidgroups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzylgroups, C₃-C₈ heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl) groups, and—CD₃;

R² is absent or a linker;

R³ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxygroups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups,C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, and —O—C(═O)—(C₁-C₆ alkyl) groups; and

R⁴, R⁵, and R⁸ are each independently chosen from hydrogen, hydroxylgroups, —O—(C₁-C₆ alkyl) groups, —O—C(═O)—(C₁-C₆ alkyl) groups, andC₁-C₆ alkyl groups;

R⁶ and R⁷ are each independently chosen from hydrogen, —O—R¹⁷,—O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₆ alkyl groups, —NR¹⁵R¹⁶, and alinker;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷; and

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, R¹⁶, and R¹⁷ are eachindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups,—NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆alkylalkoxy groups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein at least one of R⁶ and R⁷ is hydrogen; and

wherein if R² is a linker, then neither R⁶ or R⁷ is a linker, and if R⁶or R⁷ is a linker, then R² is absent.

In various other embodiments, the linker-drug (L-D) compounds disclosedherein comprise a linker-drug structure according to Formula (VII). Invarious embodiments, the present disclosure provides a linker-drug (L-D)compound of Formula (VII):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R⁹ are each independently chosen from absent, hydrogen, C₁-C₆alkyl groups, C₁-C₆ alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆alkylcarboxylic acid groups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkylgroups, benzyl groups, C₃-C₈ heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl)groups, and —CD₃;

R² is absent or a linker; R³ is chosen from hydrogen, C₁-C₆ alkylgroups, C₁-C₆ alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆alkylcarboxylic acid groups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkylgroups, benzyl groups, C₃-C₈ heterocyclyl groups, and —O—C(═O)—(C₁-C₆alkyl) groups;

R⁴, R⁵, and R⁸ are each independently chosen from hydrogen, hydroxylgroups, —O—(C₁-C₆ alkyl) groups, —O—C(═O)—(C₁-C₆ alkyl) groups, andC₁-C₆ alkyl groups;

R⁶ and R⁷ are each independently chosen from hydrogen, —O—R¹⁷,—O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₆ alkyl groups, —NR¹⁵R¹⁶, and alinker;

R¹⁰ is chosen from hydrogen, C₁-C₆ alkyl groups, —C(═O)—(C₁-C₆ alkyl)groups, and —CD₃;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷;

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups; and

a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹⁵, R¹⁶, and R¹⁷ areeach independently substituted with 0 to 3 groups independently chosenfrom halogens, hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl)groups, —NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups,C₁-C₆ alkylalkoxy groups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein at least one of R⁶ and R⁷ is hydrogen;

wherein if R² is a linker, then neither R⁶ or R⁷ is a linker, and if R⁶or R⁷ is a linker, then R² is absent; and

wherein R¹ and R⁹ cannot both be absent.

In various other embodiments, the linker-drug (L-D) compounds disclosedherein comprise a linker-drug structure according to Formula (IX). Invarious embodiments, the present disclosure provides a linker-drug (L-D)compound of Formula (IX):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is chosen from absent, hydrogen, C₁-C₆ alkyl groups, C₁-C₆alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acidgroups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzylgroups, C₃-C₈ heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl) groups, and—CD₃;

R² is a linker;

R³ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxygroups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups,C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, and —O—C(═O)—(C₁-C₆ alkyl) groups;

R⁴ is chosen from hydrogen, hydroxyl groups, —O—(C₁-C₆ alkyl) groups,—O—C(═O)—(C₁-C₆ alkyl) groups, and C₁-C₆ alkyl groups;

R¹⁰ is chosen from 3 to 10 membered carbocycles and 3 to 10 memberedheterocycles, each of which is substituted with 0 to 3 R^(a), whereineach R^(a) is independently chosen from halogens, C₁-C₆ alkyl groups,—O—(C₁-C₆)alkyl groups, C₁-C₆ alkylalkoxy groups, C₁-C₆ alkylhydroxygroups, —S(═O)_(w)-(4 to 7 membered heterocycles), 4 to 7 memberedcarbocycles, and 4 to 7 membered heterocycles;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷; and

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein R¹, R², R³, R⁴, R¹⁰, R¹⁵, R¹⁶, and R¹⁷ are each independentlysubstituted with 0 to 3 groups independently chosen from halogens,hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups, —NR¹⁵R¹⁶,C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆ alkylalkoxygroups, benzyl groups, and C₃-C₈ heterocyclyl groups; and

wherein each R^(a) is independently substituted with 0 to 3 groupsindependently chosen from halogens, hydroxyl groups, —NR¹⁵R¹⁶, C₁-C₆alkyl groups, —(C═O)—(C₁-C₆ alkyl) groups, —(C═O)—(C₁-C₆ alkyl)-(C₃-C₁₀heterocyclyl)groups, and C₁-C₆ alkylcarboxylic acid groups, each ofwhich is substituted with 0, 1, or 2 groups independently chosen fromhalogens, hydroxyl groups, —NR¹⁵R¹⁶, and C₁-C₃ alkyl groups; and

w is 0, 1, or 2.

Also, in various embodiments, provided herein are therapeutic uses forthe described ADC compounds and compositions, e.g., in treating aneoplastic disorder, e.g., a cancer. In certain aspects, the presentdisclosure provides methods of treating a neoplastic disorder, e.g., acancer that expresses an antigen targeted by the antibody or antigenbinding fragment of the ADC, such as HER2, CD138, EPHA2, MSLN, FOLH1,CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6,SLC44A4, or STEAP1.

In certain aspects, the present disclosure provides methods of treatinga subject having or suspected of having a neoplastic disorder byadministering to the subject a therapeutically effective amount and/orregimen of any one of the described ADCs or compositions. In someembodiments, the neoplastic disorder is a hematological malignancy or asolid tumor. In some embodiments, the neoplastic disorder is ahematological malignancy. In some embodiments, the hematologicalmalignancy is selected from a B-cell malignancy, a leukemia, a lymphoma,and a myeloma. In some embodiments, the hematological malignancy isselected from acute myeloid leukemia and multiple myeloma. In someembodiments, the neoplastic disorder is a solid tumor. In someembodiments, the solid tumor is selected from breast cancer (e.g.,HER2-positive breast cancer), gastric cancer (e.g., gastricadenocarcinoma), prostate cancer, ovarian cancer, lung cancer (e.g.,lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrialcarcinoma), salivary duct carcinoma, melanoma, colon cancer, cervicalcancer, pancreatic cancer, kidney cancer, colorectal cancer, andesophageal cancer. In some embodiments, the solid tumor is selected fromHER2-positive breast cancer, gastric adenocarcinoma, prostate cancer,and osteosarcoma.

In some embodiments, treatment with the antibody-drug conjugate orcomposition induces bystander killing of neoplastic cells which do notexpress a target antigen but are adjacent to neoplastic cells whichexpress a target antigen. In some embodiments, the subject has one ormore neoplastic cells which express a target antigen.

In some embodiments, the target antigen is HER2. In some embodiments,the one or more neoplastic cells are derived from a HER2-expressingbreast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lungadenocarcinoma), uterine cancer (e.g., uterine serous endometrialcarcinoma), osteosarcoma, or salivary duct carcinoma.

In some embodiments, the subject is non-responsive or poorly responsiveto treatment with (a) an anti-HER2 antibody when administered aloneand/or (b) a splicing modulator when administered alone. In someembodiments, the subject is intolerant, non-responsive, or poorlyresponsive to treatment with a splicing modulator when administeredalone.

In some embodiments, the target antigen is CD138. In some embodiments,the one or more neoplastic cells are derived from a CD138-expressingmultiple myeloma. In some embodiments, the subject is non-responsive orpoorly responsive to treatment with (a) an anti-CD138 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone. In some embodiments, the subject is intolerant, non-responsive,or poorly responsive to treatment with a splicing modulator whenadministered alone.

In some embodiments, the target antigen is EPHA2. In some embodiments,the one or more neoplastic cells are derived from an EPHA2-expressingbreast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma,colon cancer, or esophageal cancer. In some embodiments, the subject isnon-responsive or poorly responsive to treatment with (a) an anti-EPHA2antibody when administered alone and/or (b) a splicing modulator whenadministered alone. In some embodiments, the subject is intolerant,non-responsive, or poorly responsive to treatment with a splicingmodulator when administered alone.

In some embodiments, the target antigen is MSLN. In some embodiments,the one or more neoplastic cells are derived from a MSLN-expressingovarian cancer, cervical cancer, pancreatic cancer, or lung cancer(e.g., lung adenocarcinoma). In some embodiments, the subject isnon-responsive or poorly responsive to treatment with (a) an anti-MSLNantibody when administered alone and/or (b) a splicing modulator whenadministered alone. In some embodiments, the subject is intolerant,non-responsive, or poorly responsive to treatment with a splicingmodulator when administered alone.

In some embodiments, the target antigen is FOLH1. In some embodiments,the one or more neoplastic cells are derived from a FOLH1-expressingprostate cancer. In some embodiments, the subject is non-responsive orpoorly responsive to treatment with (a) an anti-FOLH1 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone. In some embodiments, the subject is intolerant, non-responsive,or poorly responsive to treatment with a splicing modulator whenadministered alone.

In some embodiments, the target antigen is CDH6. In some embodiments,the one or more neoplastic cells are derived from a CDH6-expressingkidney cancer. In some embodiments, the subject is non-responsive orpoorly responsive to treatment with (a) an anti-CDH6 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone. In some embodiments, the subject is intolerant, non-responsive,or poorly responsive to treatment with a splicing modulator whenadministered alone.

In some embodiments, the target antigen is CEACAM5. In some embodiments,the one or more neoplastic cells are derived from a CEACAM5-expressingcolorectal cancer. In some embodiments, the subject is non-responsive orpoorly responsive to treatment with (a) an anti-CEACAM5 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone. In some embodiments, the subject is intolerant, non-responsive,or poorly responsive to treatment with a splicing modulator whenadministered alone.

In some embodiments, the target antigen is CFC1B. In some embodiments,the one or more neoplastic cells are derived from a CFC1B-expressingpancreatic cancer. In some embodiments, the subject is non-responsive orpoorly responsive to treatment with (a) an anti-CFC1B antibody whenadministered alone and/or (b) a splicing modulator when administeredalone. In some embodiments, the subject is intolerant, non-responsive,or poorly responsive to treatment with a splicing modulator whenadministered alone.

In some embodiments, the target antigen is ENPP3. In some embodiments,the one or more neoplastic cells are derived from an ENPP3-expressingkidney cancer. In some embodiments, the subject is non-responsive orpoorly responsive to treatment with (a) an anti-ENPP3 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone. In some embodiments, the subject is intolerant, non-responsive,or poorly responsive to treatment with a splicing modulator whenadministered alone.

In some embodiments, the target antigen is FOLR1. In some embodiments,the one or more neoplastic cells are derived from a FOLR1-expressingovarian cancer. In some embodiments, the subject is non-responsive orpoorly responsive to treatment with (a) an anti-FOLR1 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone. In some embodiments, the subject is intolerant, non-responsive,or poorly responsive to treatment with a splicing modulator whenadministered alone.

In some embodiments, the target antigen is HAVCR1. In some embodiments,the one or more neoplastic cells are derived from a HAVCR1-expressingkidney cancer or esophageal cancer. In some embodiments, the subject isnon-responsive or poorly responsive to treatment with (a) an anti-HAVCR1antibody when administered alone and/or (b) a splicing modulator whenadministered alone. In some embodiments, the subject is intolerant,non-responsive, or poorly responsive to treatment with a splicingmodulator when administered alone.

In some embodiments, the target antigen is KIT. In some embodiments, theone or more neoplastic cells are derived from a KIT-expressing kidneycancer. In some embodiments, the subject is non-responsive or poorlyresponsive to treatment with (a) an anti-KIT antibody when administeredalone and/or (b) a splicing modulator when administered alone. In someembodiments, the subject is intolerant, non-responsive, or poorlyresponsive to treatment with a splicing modulator when administeredalone.

In some embodiments, the target antigen is MET. In some embodiments, theone or more neoplastic cells are derived from a MET-expressing kidneycancer or esophageal cancer. In some embodiments, the subject isnon-responsive or poorly responsive to treatment with (a) an anti-METantibody when administered alone and/or (b) a splicing modulator whenadministered alone. In some embodiments, the subject is intolerant,non-responsive, or poorly responsive to treatment with a splicingmodulator when administered alone.

In some embodiments, the target antigen is MUC16. In some embodiments,the one or more neoplastic cells are derived from a MUC16-expressingovarian cancer, cervical cancer, or breast cancer. In some embodiments,the subject is non-responsive or poorly responsive to treatment with (a)an anti-MUC16 antibody when administered alone and/or (b) a splicingmodulator when administered alone. In some embodiments, the subject isintolerant, non-responsive, or poorly responsive to treatment with asplicing modulator when administered alone.

In some embodiments, the target antigen is SLC39A6. In some embodiments,the one or more neoplastic cells are derived from a SLC39A6-expressingbreast cancer or prostate cancer. In some embodiments, the subject isnon-responsive or poorly responsive to treatment with (a) ananti-SLC39A6 antibody when administered alone and/or (b) a splicingmodulator when administered alone. In some embodiments, the subject isintolerant, non-responsive, or poorly responsive to treatment with asplicing modulator when administered alone.

In some embodiments, the target antigen is SLC44A4. In some embodiments,the one or more neoplastic cells are derived from a SLC44A4-expressingprostate cancer. In some embodiments, the subject is non-responsive orpoorly responsive to treatment with (a) an anti-SLC44A4 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone. In some embodiments, the subject is intolerant, non-responsive,or poorly responsive to treatment with a splicing modulator whenadministered alone.

In some embodiments, the target antigen is STEAP1. In some embodiments,the one or more neoplastic cells are derived from a STEAP1-expressingprostate cancer. In some embodiments, the subject is non-responsive orpoorly responsive to treatment with (a) an anti-STEAP1 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone. In some embodiments, the subject is intolerant, non-responsive,or poorly responsive to treatment with a splicing modulator whenadministered alone.

In certain other aspects, the present disclosure provides methods ofreducing or inhibiting growth of a tumor in a subject having orsuspected of having a neoplastic disorder by administering to thesubject a therapeutically effective amount and/or regimen of any one ofthe described ADCs or compositions.

In some embodiments, treatment with the antibody-drug conjugate orcomposition induces bystander killing of neoplastic tumor cells which donot express a target antigen but are adjacent to neoplastic tumor cellswhich express a target antigen. In some embodiments, the tumor comprisesone or more neoplastic cells which express a target antigen.

In some embodiments, the target antigen is HER2. In some embodiments,the one or more neoplastic cells are derived from a HER2-expressingbreast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lungadenocarcinoma), uterine cancer (e.g., uterine serous endometrialcarcinoma), osteosarcoma, or salivary duct carcinoma. In someembodiments, the tumor is resistant or refractory to treatment with (a)an anti-HER2 antibody when administered alone and/or (b) a splicingmodulator when administered alone.

In some embodiments, the target antigen is CD138. In some embodiments,the one or more neoplastic cells are derived from a CD138-expressingmultiple myeloma. In some embodiments, the tumor is resistant orrefractory to treatment with (a) an anti-CD138 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone.

In some embodiments, the target antigen is EPHA2. In some embodiments,the one or more neoplastic cells are derived from an EPHA2-expressingbreast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma,colon cancer, or esophageal cancer. In some embodiments, the tumor isresistant or refractory to treatment with (a) an anti-EPHA2 antibodywhen administered alone and/or (b) a splicing modulator whenadministered alone.

In some embodiments, the target antigen is MSLN. In some embodiments,the one or more neoplastic cells are derived from a MSLN-expressingovarian cancer, cervical cancer, pancreatic cancer, or lung cancer(e.g., lung adenocarcinoma). In some embodiments, the tumor is resistantor refractory to treatment with (a) an anti-MSLN antibody whenadministered alone and/or (b) a splicing modulator when administeredalone.

In some embodiments, the target antigen is FOLH1. In some embodiments,the one or more neoplastic cells are derived from a FOLH1-expressingprostate cancer. In some embodiments, the tumor is resistant orrefractory to treatment with (a) an anti-FOLH1 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone.

In some embodiments, the target antigen is CDH6. In some embodiments,the one or more neoplastic cells are derived from a CDH6-expressingkidney cancer. In some embodiments, the tumor is resistant or refractoryto treatment with (a) an anti-CDH6 antibody when administered aloneand/or (b) a splicing modulator when administered alone.

In some embodiments, the target antigen is CEACAM5. In some embodiments,the one or more neoplastic cells are derived from a CEACAM5-expressingcolorectal cancer. In some embodiments, the tumor is resistant orrefractory to treatment with (a) an anti-CEACAM5 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone.

In some embodiments, the target antigen is CFC1B. In some embodiments,the one or more neoplastic cells are derived from a CFC1B-expressingpancreatic cancer. In some embodiments, the tumor is resistant orrefractory to treatment with (a) an anti-CFC1B antibody whenadministered alone and/or (b) a splicing modulator when administeredalone.

In some embodiments, the target antigen is ENPP3. In some embodiments,the one or more neoplastic cells are derived from an ENPP3-expressingkidney cancer. In some embodiments, the tumor is resistant or refractoryto treatment with (a) an anti-ENPP3 antibody when administered aloneand/or (b) a splicing modulator when administered alone.

In some embodiments, the target antigen is FOLR1. In some embodiments,the one or more neoplastic cells are derived from a FOLR1-expressingovarian cancer. In some embodiments, the tumor is resistant orrefractory to treatment with (a) an anti-FOLR1 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone.

In some embodiments, the target antigen is HAVCR1. In some embodiments,the one or more neoplastic cells are derived from a HAVCR1-expressingkidney cancer or esophageal cancer. In some embodiments, the tumor isresistant or refractory to treatment with (a) an anti-HAVCR1 antibodywhen administered alone and/or (b) a splicing modulator whenadministered alone.

In some embodiments, the target antigen is KIT. In some embodiments, theone or more neoplastic cells are derived from a KIT-expressing kidneycancer. In some embodiments, the tumor is resistant or refractory totreatment with (a) an anti-KIT antibody when administered alone and/or(b) a splicing modulator when administered alone.

In some embodiments, the target antigen is MET. In some embodiments, theone or more neoplastic cells are derived from a MET-expressing kidneycancer or esophageal cancer. In some embodiments, the tumor is resistantor refractory to treatment with (a) an anti-MET antibody whenadministered alone and/or (b) a splicing modulator when administeredalone.

In some embodiments, the target antigen is MUC16. In some embodiments,the one or more neoplastic cells are derived from a MUC16-expressingovarian cancer, cervical cancer, or breast cancer. In some embodiments,the tumor is resistant or refractory to treatment with (a) an anti-MUC16antibody when administered alone and/or (b) a splicing modulator whenadministered alone.

In some embodiments, the target antigen is SLC39A6. In some embodiments,the one or more neoplastic cells are derived from a SLC39A6-expressingbreast cancer or prostate cancer. In some embodiments, the tumor isresistant or refractory to treatment with (a) an anti-SLC39A6 antibodywhen administered alone and/or (b) a splicing modulator whenadministered alone.

In some embodiments, the target antigen is SLC44A4. In some embodiments,the one or more neoplastic cells are derived from a SLC44A4-expressingprostate cancer. In some embodiments, the tumor is resistant orrefractory to treatment with (a) an anti-SLC44A4 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone.

In some embodiments, the target antigen is STEAP1. In some embodiments,the one or more neoplastic cells are derived from a STEAP1-expressingprostate cancer. In some embodiments, the tumor is resistant orrefractory to treatment with (a) an anti-STEAP1 antibody whenadministered alone and/or (b) a splicing modulator when administeredalone.

In still other aspects, the present disclosure provides methods ofdetermining whether a subject having or suspected of having a neoplasticdisorder will be responsive to treatment with any one of the describedADCs or compositions by providing a biological sample from the subjectand contacting the biological sample with the ADC or composition. Insome embodiments, the biological sample is a tumor sample. In someembodiments, the tumor sample is a tumor biopsy or blood sample. In someembodiments, the blood sample is selected from blood, a blood fraction,or a cell obtained from the blood or blood fraction. In someembodiments, the subject has one or more neoplastic cells which expressa target antigen. In some embodiments, the target antigen is HER2. Insome embodiments, the one or more neoplastic cells are derived from aHER2-expressing breast cancer, ovarian cancer, gastric cancer, lungcancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serousendometrial carcinoma), osteosarcoma, or salivary duct carcinoma. Insome embodiments, the target antigen is CD138. In some embodiments, theone or more neoplastic cells are derived from a CD138-expressingmultiple myeloma. In some embodiments, the target antigen is EPHA2. Insome embodiments, the one or more neoplastic cells are derived from anEPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lungcancer, melanoma, colon cancer, or esophageal cancer. In someembodiments, the target antigen is MSLN. In some embodiments, the one ormore neoplastic cells are derived from a MSLN-expressing ovarian cancer,cervical cancer, pancreatic cancer, or lung cancer (e.g., lungadenocarcinoma). In some embodiments, the target antigen is FOLH1. Insome embodiments, the one or more neoplastic cells are derived from aFOLH1-expressing prostate cancer. In some embodiments, the targetantigen is CDH6. In some embodiments, the one or more neoplastic cellsare derived from a CDH6-expressing kidney cancer. In some embodiments,the target antigen is CEACAM5. In some embodiments, the one or moreneoplastic cells are derived from a CEACAM5-expressing colorectalcancer. In some embodiments, the target antigen is CFC1B. In someembodiments, the one or more neoplastic cells are derived from aCFC1B-expressing pancreatic cancer. In some embodiments, the targetantigen is ENPP3. In some embodiments, the one or more neoplastic cellsare derived from an ENPP3-expressing kidney cancer. In some embodiments,the target antigen is FOLR1. In some embodiments, the one or moreneoplastic cells are derived from a FOLR1-expressing ovarian cancer. Insome embodiments, the target antigen is HAVCR1. In some embodiments, theone or more neoplastic cells are derived from a HAVCR1-expressing kidneycancer or esophageal cancer. In some embodiments, the target antigen isKIT. In some embodiments, the one or more neoplastic cells are derivedfrom a KIT-expressing kidney cancer. In some embodiments, the targetantigen is MET. In some embodiments, the one or more neoplastic cellsare derived from a MET-expressing kidney cancer or esophageal cancer. Insome embodiments, the target antigen is MUC16. In some embodiments, theone or more neoplastic cells are derived from a MUC16-expressing ovariancancer, cervical cancer, or breast cancer. In some embodiments, thetarget antigen is SLC39A6. In some embodiments, the one or moreneoplastic cells are derived from a SLC39A6-expressing breast cancer orprostate cancer. In some embodiments, the target antigen is SLC44A4. Insome embodiments, the one or more neoplastic cells are derived from aSLC44A4-expressing prostate cancer. In some embodiments, the targetantigen is STEAP1. In some embodiments, the one or more neoplastic cellsare derived from a STEAP1-expressing prostate cancer.

Further provided herein, in various embodiments, are pharmaceuticalcompositions comprising an ADC and a pharmaceutically acceptablediluent, carrier, and/or excipient. Methods of producing the describedADC compounds and compositions are also disclosed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows dose response of exemplary payload compounds in acompetitive binding assay. Nuclear extracts from 293F cellsoverexpressing wild type flag-tagged SF3B1 were immunoprecipitated withan anti-SF3B1 antibody and scintillation proximity assay (SPA) beadcocktail. Binding reactions contained the antibody-bead mixture andincreasing concentrations of compound, followed by competition with an³H-labelled pladienolide B (PB) probe. The y-axis represents the percentchange (% response) of specific binding relative to the DMSO control(0%). Data are represented as mean±standard deviation (SD).

FIG. 2 shows modulation of splicing by exemplary payload compounds in anin vitro splicing assay. Nuclear extracts from HeLa S3 cells wereincubated with Ad2.2 pre-mRNA and increasing concentrations of compound,followed by quantification of splicing modulation via RT-PCR. The Ad2.2sequence is derived from the adenoviral Ad2 pre-mRNA substrate withmodifications around the branch point sequence. The y-axis representsthe percent change (% response) of splicing relative to the DMSO control(0%). Data are represented as mean±SD.

FIG. 3 shows viability dose response of exemplary payload compounds inHER2-amplified breast cancer cells (HCC1954). Cells were incubated withcompound for 144 hours (6 days) and viability was read in CellTiter-Glo®reagent. Data are represented as mean±SD.

FIG. 4 shows the results of a cell binding assay. Binding of exemplaryHER2-ADCs to JIMT1 cells was assessed by flow cytometry. Meanfluorescence intensity values were measured to determine binding ofconjugates, followed by PE-labelled secondary. Data are represented asmean±SD.

FIG. 5A shows viability dose response of exemplary HER2-ADCs inHER2-amplified breast cancer cells (HCC1954). FIG. 5B shows viabilitydose response of exemplary HER2-ADCs in HER2-amplified gastric cancercells (N87). FIG. 5C shows viability dose response of exemplaryHER2-ADCs in HER2-amplified breast cancer cells (SKBR3). Cells wereincubated with conjugates for 144 hours (6 days) and viability was readin CellTiter-Glo® reagent. Data are represented as mean±SD.

FIG. 6 shows viability dose response of exemplary HER2-ADCs in non-HER2expressing breast cancer cells (MCF7). Cells were incubated withconjugates for 144 hours (6 days) and viability was read inCellTiter-Glo® reagent. Data are represented as mean±SD.

FIG. 7 shows the results of a SLC25A19 splicing assay in HER2-amplifiedbreast cancer cells (HCC1954). Cells were incubated with conjugates for24 hours and splicing of SLC25A19 transcript was measured in a real timeqPCR reaction with a specific Taqman primer-probe set. The y-axisrepresents the percent (%) response relative to the DMSO control (0%).Data are represented as mean±SD.

FIG. 8 shows the results of a bystander killing assay. H1568 cellsoverexpressing HER2 (target-positive) or H1568 cells tagged withluciferase (target-negative) plated either alone or incubated togetherin co-culture for 144 hours (6 days) were treated with exemplaryHER2-ADCs. Plates were read with OneGlo® reagent. The y-axis representsthe percent (%) response relative to the PBS control (100%). Data arerepresented as mean±SD.

FIG. 9 shows tumor growth kinetics for each group of HCC1954-implantedCB17-SCID mice treated with a single intravenous dose of an exemplaryHER2-ADC or corresponding dose-matched payload (6-10 animals per group).Tumor volumes were measured twice weekly after treatment. Data arerepresented as mean±standard error of the mean (SEM).

FIG. 10 shows viability dose response of exemplary CD138-ADCs in aCD138-expressing multiple myeloma cell line. MOLP8 cells were incubatedwith conjugates for 144 hours (6 days) and viability was read inCellTiter-Glo® reagent. Data are represented as mean±SD.

FIG. 11 shows viability dose response of exemplary EPH2A-ADCs in anEPHA2-expressing prostate cancer cell line. PC3 cells were incubatedwith conjugates for 144 hours (6 days) and viability was read inCellTiter-Glo® reagent. Data are represented as mean±SD.

FIG. 12A and FIG. 12B show the results of an in vitro stability assayfor an exemplary anti-HER2 ADC, AB185-ADL1-D1. The y-axis representsconcentration of total antibody (FIG. 12A) and conjugated (intact)payload (FIG. 12B); the x-axis represents time as measured in hours at37° C.

FIG. 13 shows plasma concentrations of an exemplary anti-HER2 ADC,AB185-ADL1-D1, following a single intravenous dose in CD17-SCID N87tumor-bearing mice.

FIG. 14 shows a schematic diagram of an exemplary RNA sequencing andprotein ligandome experiment.

FIG. 15 shows a schematic diagram of an exemplary T-cell primingexperiment.

FIG. 16 shows the results of a FACS analysis. Monocytes were isolatedfrom peripheral blood mononuclear cells (PBMC) and were induced todifferentiate into dendritic cells (DC) through culturing in a cytokinecocktail. FACS was performed to validate the maturation of DC frommonocytes.

FIG. 17A-D show the results of an ELISpot assay. FIG. 17A shows ELISpotplates indicating Neoantigen 1 priming of CD8+ T-cell activation.Stimulation of CD8+ T-cells was monitored by secretion of IFNγ. FIG. 17Bshows quantification of IFNγ spots (spot number) in Neoantigen 1 ELISpotplates (FIG. 17A). FIG. 17C shows ELISpot plates indicating Neoantigen 3priming of CD8+ T-cell activation. Stimulation of CD8+ T-cells wasmonitored by secretion of IFNγ. FIG. 17D shows quantification of IFNγspots (fold change) in Neoantigen 3 ELISpot plates (FIG. 17C).

FIG. 18 shows a plot comparing splicing potency (IC50 qPCR) againstcellular potency (GI50 CTG) for exemplary anti-HER2 ADCs in HCC1954breast cancer cells. Values shown are sized by cell lethality and shadedby depth of alternative splicing response.

FIG. 19 shows a plot comparing splicing potency (1050 qPCR) againstcellular potency (G150 CTG) for exemplary anti-HER2 ADCs in N87 gastriccancer cells. Values shown are sized by cell lethality and shaded bydepth of alternative splicing response.

FIG. 20 shows a plot comparing potency and lethality of exemplaryanti-HER2 ADCs (in HCC1954 breast cancer cells) against stability andpermeability of corresponding payloads. Values shown are sized bypayload stability and shaded by payload permeability.

FIG. 21 shows tumor growth kinetics for each group of N87-implantedCB17-SCID mice treated intravenously with vehicle or 10 mg/kgtrastuzumab, TDM1, or an exemplary HER2-ADC Q7D for 2 cycles (N=8 pergroup). Data are represented as mean±standard error of the mean (SEM)(mm³).

FIG. 22 shows body weight change for each group of N87-implantedCB17-SCID mice treated intravenously with vehicle or 10 mg/kgtrastuzumab, TDM1, or an exemplary HER2-ADC Q7D for 2 cycles (N=8 pergroup). Data are represented as mean±standard error of the mean (SEM)(%).

FIG. 23 shows tumor growth kinetics (left) and body weight change(right) for each group of N87-implanted CB17-SCID mice treatedintravenously with vehicle or 10 mg/kg trastuzumab, TDM1, or anexemplary HER2-ADC Q7D for 2 cycles (N=8 per group). Data arerepresented as mean±SEM (tumor volume, mm³) or mean±SEM (body weight,%).

FIG. 24A-24D show pharmacodynamics (PD) modulation of mRNA junctions inN87-implanted CB17-SCID mice treated intravenously with vehicle or 10mg/kg trastuzumab, TDM1, or an exemplary HER2-ADC Q7D for 2 cycles.RT-qPCR of FBXW5 (mature mRNA transcript) was monitored and is shown inFIG. 24A and FIG. 24C. RT-qPCR of TAOK1 (neojunction transcript) wasmonitored and is shown in FIG. 24B and FIG. 24D. Animals (N=4 per group)were collected at either 48 hours (FIG. 24A and FIG. 24B) or at thetimes indicated (FIG. 24C and FIG. 24D). Tumors were isolated for RNAextraction and RT-qPCR.

FIG. 25 shows a schematic diagram of an exemplary target indicationanalysis.

FIG. 26 shows an exemplary bioconjugation scheme for preparation of ADCsusing splicing modulators.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosed compositions and methods may be understood more readily byreference to the following detailed description taken in connection withthe accompanying figures, which form a part of this disclosure.

Throughout this text, the descriptions refer to compositions and methodsof using the compositions. Where the disclosure describes or claims afeature or embodiment associated with a composition, such a feature orembodiment is equally applicable to the methods of using thecomposition. Likewise, where the disclosure describes or claims afeature or embodiment associated with a method of using a composition,such a feature or embodiment is equally applicable to the composition.

When a range of values is expressed, it includes embodiments using anyparticular value within the range. Further, reference to values statedin ranges includes each and every value within that range. All rangesare inclusive of their endpoints and combinable. When values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment.Reference to a particular numerical value includes at least thatparticular value, unless the context clearly dictates otherwise. The useof “or” will mean “and/or” unless the specific context of its usedictates otherwise. All references cited herein are incorporated byreference for any purpose. Where a reference and the specificationconflict, the specification will control.

It is to be appreciated that certain features of the disclosedcompositions and methods, which are, for clarity, described herein inthe context of separate embodiments, may also be provided in combinationin a single embodiment. Conversely, various features of the disclosedcompositions and methods that are, for brevity, described in the contextof a single embodiment, may also be provided separately or in anysubcombination.

Definitions

Various terms relating to aspects of the description are used throughoutthe specification and claims. Such terms are to be given their ordinarymeaning in the art unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinitions provided herein.

As used herein, the singular forms “a,” “an,” and “the” include pluralforms unless the context clearly dictates otherwise.

The terms “about” or “approximately” in the context of numerical valuesand ranges refers to values or ranges that approximate or are close tothe recited values or ranges such that the embodiment may perform asintended, such as having a desired amount of nucleic acids orpolypeptides in a reaction mixture, as is apparent to the skilled personfrom the teachings contained herein. In some embodiments, about meansplus or minus 10% of a numerical amount.

The terms “antibody-drug conjugate,” “antibody conjugate,” “conjugate,”“immunoconjugate,” and “ADC” are used interchangeably, and refer to oneor more therapeutic compounds (e.g., a splicing modulator) that islinked to one or more antibodies or antigen binding fragments and isdefined by the generic formula: Ab-(L-D)_(p) (Formula I), wherein Ab=anantibody or antigen binding fragment, L=a linker moiety, D=a drug moiety(e.g., a splicing modulator drug moiety), and p=the number of drugmoieties per antibody or antigen binding fragment. An ADC comprising asplicing modulator drug moiety may also be referred to herein morespecifically as a “splicing modulator-loaded antibody” or a “SMLA.” InADCs comprising a splicing modulator drug moiety, “p” refers to thenumber of splicing modulator compounds linked to the antibody or antigenbinding fragment. In some embodiments, the linker L can include acleavable moiety between the antibody or antigen binding fragment andthe therapeutic compound. In some embodiments, the linker L can includea cleavable moiety that can be attached to either or both the antibodyor antigen binding fragment and therapeutic compound by spacer unit(s).In some embodiments, when a spacer unit attaches the cleavable moiety tothe therapeutic compound, it is a self-immolative spacer unit. In otherembodiments, the linker L does not include a cleavable moiety, and is anon-cleavable linker. In some embodiments, the linker L can include atleast one spacer unit that can directly attach to the antibody orantigen binding fragment and to the therapeutic compound. Exemplarycleavable and non-cleavable linkers are described and exemplifiedherein.

The term “antibody” is used in the broadest sense to refer to animmunoglobulin molecule that recognizes and specifically binds to atarget, such as a protein, polypeptide, carbohydrate, polynucleotide,lipid, or combinations of the foregoing through at least one antigenrecognition site within the variable region of the immunoglobulinmolecule. The heavy chain of an antibody is composed of a heavy chainvariable domain (V_(H)) and a heavy chain constant region (C_(H)). Thelight chain is composed of a light chain variable domain (V_(L)) and alight chain constant domain (CO. For the purposes of this application,the mature heavy chain and light chain variable domains each comprisethree complementarity determining regions (CDR1, CDR2 and CDR3) withinfour framework regions (FR1, FR2, FR3, and FR4) arranged from N-terminusto C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. An “antibody”can be naturally occurring or man-made, such as monoclonal antibodiesproduced by conventional hybridoma technology. The term “antibody”includes full-length monoclonal antibodies and full-length polyclonalantibodies, as well as antibody fragments such as Fab, Fab′, F(ab′)₂,Fv, and single chain antibodies. An antibody can be any one of the fivemajor classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, orsubclasses thereof (e.g., isotypes IgG1, IgG2, IgG3, IgG4). The termfurther encompasses human antibodies, chimeric antibodies, humanizedantibodies and any modified immunoglobulin molecule containing anantigen recognition site, so long as it demonstrates the desiredbiological activity (e.g., binds the target antigen, internalizes withina target-antigen expressing cell).

The term “monoclonal antibody,” as used herein, refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic epitope. In contrast, conventional(polyclonal) antibody preparations typically include a multitude ofantibodies directed against (or specific for) different epitopes. Themodifier “monoclonal” indicates the character of the antibody as beingobtained from a substantially homogeneous population of antibodies, andis not to be construed as requiring production of the antibody by anyparticular method. For example, the monoclonal antibodies to be used inaccordance with the present disclosure may be made by the hybridomamethod first described by Kohler et al. (1975) Nature 256:495, or may bemade by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).Monoclonal antibodies may also be isolated from phage antibody librariesusing the techniques described in Clackson et al. (1991) Nature352:624-8, and Marks et al. (1991) J Mol Biol. 222:581-97, for example.

The monoclonal antibodies described herein specifically include“chimeric” antibodies, in which a portion of the heavy and/or lightchain is identical with or homologous to corresponding sequences inantibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey specifically bind the target antigen and/or exhibit the desiredbiological activity.

The term “human antibody,” as used herein, refers an antibody producedby a human or an antibody having an amino acid sequence of an antibodyproduced by a human.

The term “chimeric antibody,” as used herein, refers to antibodieswherein the amino acid sequence of the immunoglobulin molecule isderived from two or more species. In some instances, the variableregions of both heavy and light chains correspond to the variableregions of antibodies derived from one species with the desiredspecificity, affinity, and activity while the constant regions arehomologous to antibodies derived from another species (e.g., human) tominimize an immune response in the latter species.

As used herein, the term “humanized antibody” refers to forms ofantibodies that contain sequences from non-human (e.g., murine)antibodies as well as human antibodies. Such antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe framework (FR) regions are those of a human immunoglobulin sequence.The humanized antibody optionally also will comprise at least a portionof an immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. The humanized antibody can be further modified by thesubstitution of residues, either in the Fv framework region and/orwithin the replaced non-human residues to refine and optimize antibodyspecificity, affinity, and/or activity.

The term “antigen binding fragment” or “antigen binding portion” of anantibody, as used herein, refers to one or more fragments of an antibodyor protein that retain the ability to specifically bind to an antigen(e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3,FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, STEAP1). Antigenbinding fragments may also retain the ability to internalize into anantigen-expressing cell. In some embodiments, antigen binding fragmentsalso retain immune effector activity. It has been shown that fragmentsof a full-length antibody can perform the antigen binding function of afull-length antibody. Examples of binding fragments encompassed withinthe term “antigen binding fragment” or “antigen binding portion” of anantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe V_(L), V_(H), C_(L), and C_(H1) domains; (ii) a F(ab′)₂ fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region; (iii) a Fd fragment consisting of the V_(H)and C_(H1) domains; (iv) a Fv fragment consisting of the V_(L) and V_(H)domains of a single arm of an antibody; (v) a dAb fragment, whichcomprises a single variable domain, e.g., a V_(H) domain (see, e.g.,Ward et al. (1989) Nature 341:544-6; and Intl. Pub. No. WO 1990/005144);and (vi) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, V_(L) andV_(H), are coded for by separate genes, they can be joined, usingrecombinant methods, by a synthetic linker that enables them to be madeas a single protein chain in which the V_(L) and V_(H) regions pair toform monovalent molecules (known as single chain Fv (scFv)). See, e.g.,Bird et al. (1988) Science 242:423-6; and Huston et al. (1988) Proc NatlAcad Sci. USA 85:5879-83. Such single chain antibodies are also intendedto be encompassed within the term “antigen binding fragment” or “antigenbinding portion” of an antibody, and are known in the art as anexemplary type of binding fragment that can internalize into cells uponbinding (see, e.g., Zhu et al. (2010) 9:2131-41; He et al. (2010) J NuclMed. 51:427-32; and Fitting et al. (2015) MAbs 7:390-402). In certainembodiments, scFv molecules may be incorporated into a fusion protein.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in whichV_(H) and V_(L) domains are expressed on a single polypeptide chain, butusing a linker that is too short to allow for pairing between the twodomains on the same chain, thereby forcing the domains to pair withcomplementary domains of another chain and creating two antigen bindingsites (see e.g., Holliger et al. (1993) Proc Natl Acad Sci. USA90:6444-8; and Poljak et al. (1994) Structure 2:1121-3). Antigen bindingfragments are obtained using conventional techniques known to those ofskill in the art, and the binding fragments are screened for utility(e.g., binding affinity, internalization) in the same manner as areintact antibodies. Antigen binding fragments may be prepared by cleavageof the intact protein, e.g., by protease or chemical cleavage.

“Internalizing” as used herein in reference to an antibody or antigenbinding fragment refers to an antibody or antigen binding fragment thatis capable of being taken through the cell's lipid bilayer membrane toan internal compartment (i.e., “internalized”) upon binding to the cell,preferably into a degradative compartment in the cell. For example, aninternalizing anti-HER2 antibody is one that is capable of being takeninto the cell after binding to HER2 on the cell membrane. In someembodiments, the antibody or antigen binding fragment used in the ADCsdisclosed herein targets a cell surface antigen (e.g., HER2) and is aninternalizing antibody or internalizing antigen binding fragment (i.e.,the ADC transfers through the cellular membrane after antigen binding).In some embodiments, the internalizing antibody or antigen bindingfragment binds a receptor on the cell surface. An internalizing antibodyor internalizing antigen binding fragment that targets a receptor on thecell membrane may induce receptor-mediated endocytosis. In someembodiments, the internalizing antibody or internalizing antigen bindingfragment is taken into the cell via receptor-mediated endocytosis.

“Non-internalizing” as used herein in reference to an antibody orantigen binding fragment refers to an antibody or antigen bindingfragment that remains at the cell surface upon binding to the cell. Insome embodiments, the antibody or antigen binding fragment used in theADCs disclosed herein targets a cell surface antigen and is anon-internalizing antibody or non-internalizing antigen binding fragment(i.e., the ADC remains at the cell surface and does not transfer throughthe cellular membrane after antigen binding). In some embodiments, thenon-internalizing antibody or antigen binding fragment binds anon-internalizing receptor or other cell surface antigen. Exemplarynon-internalizing cell surface antigens include but are not limited toCA125 and CEA, and antibodies that bind to non-internalizing antigentargets are also known in the art (see, e.g., Bast et al. (1981) J ClinInvest. 68(5):1331-7; Scholler and Urban (2007) Biomark Med.1(4):513-23; and Boudousq et al. (2013) PLoS One 8(7):e69613).

The term “human epidermal growth factor receptor 2,” “HER2,” or“HER2/NEU,” as used herein, refers to any native form of human HER2. Theterm encompasses full-length HER2 (e.g., UniProt Reference Sequence:P04626; SEQ ID NO:31), as well as any form of human HER2 that may resultfrom cellular processing. The term also encompasses functional variantsor fragments of human HER2, including but not limited to splicevariants, allelic variants, and isoforms that retain one or morebiologic functions of human HER2 (i.e., variants and fragments areencompassed unless the context indicates that the term is used to referto the wild-type protein only). HER2 can be isolated from human, or maybe produced recombinantly or by synthetic methods.

The term “anti-HER2 antibody” or “antibody that binds to HER2” refers toany form of antibody or fragment thereof that binds, e.g., specificallybinds, to HER2, and encompasses monoclonal antibodies (includingfull-length monoclonal antibodies), polyclonal antibodies, andbiologically functional antibody fragments so long as they bind, e.g.,specifically bind, to HER2. U.S. Pat. No. 5,821,337 provides and isincorporated herein by reference for exemplary HER2-binding sequences,including exemplary anti-HER2 antibody sequences. In some embodiments,the anti-HER2 antibody used in the ADCs disclosed herein is aninternalizing antibody or internalizing antibody fragment. Trastuzumab(U.S. Pat. No. 5,821,337; Molina et al. (2001) Cancer Res.61(12):4744-9) is an exemplary anti-human HER2 antibody.

The term “syndecan-1,” “SDC1,” or “CD138,” as used herein, refers to anynative form of human CD138. The term encompasses full-length CD138(e.g., UniProt Reference Sequence: P18827; SEQ ID NO:32), as well as anyform of human CD138 that may result from cellular processing. The termalso encompasses functional variants or fragments of human CD138,including but not limited to splice variants, allelic variants, andisoforms that retain one or more biologic functions of human CD138(i.e., variants and fragments are encompassed unless the contextindicates that the term is used to refer to the wild-type protein only).CD138 can be isolated from a human, or may be produced recombinantly orby synthetic methods.

The term “anti-CD138 antibody” or “antibody that binds to CD138” refersto any form of antibody or fragment thereof that binds, e.g.,specifically binds, to CD138, and encompasses monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,and biologically functional antibody fragments so long as they bind,e.g., specifically bind, to CD138. In some embodiments, the anti-CD138antibody used in the ADCs disclosed herein is an internalizing antibodyor internalizing antibody fragment. B-B4 (Tassone et al. (2004) Blood104:3688-96) is an exemplary anti-human CD138 antibody.

The term “ephrin type-A receptor 2” or “EPHA2,” as used herein, refersto any native form of human EPHA2. The term encompasses full-lengthEPHA2 (e.g., UniProt Reference Sequence: P29317; SEQ ID NO:33), as wellas any form of human EPHA2 that may result from cellular processing. Theterm also encompasses functional variants or fragments of human EPHA2,including but not limited to splice variants, allelic variants, andisoforms that retain one or more biologic functions of human EPHA2(i.e., variants and fragments are encompassed unless the contextindicates that the term is used to refer to the wild-type protein only).EPHA2 can be isolated from a human, or may be produced recombinantly orby synthetic methods.

The term “anti-EPHA2 antibody” or “antibody that binds to EPHA2” refersto any form of antibody or fragment thereof that binds, e.g.,specifically binds, to EPHA2, and encompasses monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,and biologically functional antibody fragments so long as they bind,e.g., specifically bind, to EPHA2. WO 2007/030642 provides and isincorporated herein by reference for exemplary EPHA2-binding sequences,including exemplary anti-EPHA2 antibody sequences. In some embodiments,the anti-EPHA2 antibody used in the ADCs disclosed herein is aninternalizing antibody or internalizing antibody fragment. 1C1 (WO2007/030642; Jackson et al. (2008) Cancer Res. 68(22): 9367-74) is anexemplary anti-human EPHA2 antibody.

The term “mesothelin” or “MSLN,” as used herein, refers to any nativeform of human MSLN. The term encompasses full-length MSLN (e.g., UniProtReference Sequence: Q13421; SEQ ID NO:43), as well as any form of humanMSLN that may result from cellular processing. The term also encompassesfunctional variants or fragments of human MSLN, including but notlimited to splice variants, allelic variants, and isoforms that retainone or more biologic functions of human MSLN (i.e., variants andfragments are encompassed unless the context indicates that the term isused to refer to the wild-type protein only). MSLN can be isolated froma human, or may be produced recombinantly or by synthetic methods.

The term “anti-MSLN antibody” or “antibody that binds to MSLN” refers toany form of antibody or fragment thereof that binds, e.g., specificallybinds, to MSLN, and encompasses monoclonal antibodies (includingfull-length monoclonal antibodies), polyclonal antibodies, andbiologically functional antibody fragments so long as they bind, e.g.,specifically bind, to MSLN. WO 2011/074621 provides and is incorporatedherein by reference for exemplary MSLN-binding sequences, includingexemplary anti-MSLN antibody sequences. In some embodiments, theanti-MSLN antibody used in the ADCs disclosed herein is an internalizingantibody or internalizing antibody fragment. 11-25, IC14-30, IC7-4,IC17-35 and 2-9 are exemplary anti-human MSLN antibodies.

The term “glutamate carboxypeptidase 2” or “FOLH1,” as used herein,refers to any native form of human FOLH1. The term encompassesfull-length FOLH1 (e.g., UniProt Reference Sequence: Q04609; SEQ IDNO:44), as well as any form of human FOLH1 that may result from cellularprocessing. The term also encompasses functional variants or fragmentsof human FOLH1, including but not limited to splice variants, allelicvariants, and isoforms that retain one or more biologic functions ofhuman FOLH1 (i.e., variants and fragments are encompassed unless thecontext indicates that the term is used to refer to the wild-typeprotein only). FOLH1 can be isolated from a human, or may be producedrecombinantly or by synthetic methods.

The term “anti-FOLH1 antibody” or “antibody that binds to FOLH1” refersto any form of antibody or fragment thereof that binds, e.g.,specifically binds, to FOLH1, and encompasses monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,and biologically functional antibody fragments so long as they bind,e.g., specifically bind, to FOLH1. WO 2019/012260 and WO 2017/212250provide and are incorporated herein by reference for exemplaryFOLH1-binding sequences, including exemplary anti-FOLH1 antibodysequences. In some embodiments, the anti-FOLH1 antibody used in the ADCsdisclosed herein is an internalizing antibody or internalizing antibodyfragment. J591 (deimmunized) is an exemplary anti-human FOLH1 antibody.

The term “cadherin-6” or “CDH6,” as used herein, refers to any nativeform of human CDH6. The term encompasses full-length CDH6 (e.g., UniProtReference Sequence: P55285; SEQ ID NO:45), as well as any form of humanCDH6 that may result from cellular processing. The term also encompassesfunctional variants or fragments of human CDH6, including but notlimited to splice variants, allelic variants, and isoforms that retainone or more biologic functions of human CDH6 (i.e., variants andfragments are encompassed unless the context indicates that the term isused to refer to the wild-type protein only). CDH6 can be isolated froma human, or may be produced recombinantly or by synthetic methods.

The term “anti-CDH6 antibody” or “antibody that binds to CDH6” refers toany form of antibody or fragment thereof that binds, e.g., specificallybinds, to CDH6, and encompasses monoclonal antibodies (includingfull-length monoclonal antibodies), polyclonal antibodies, andbiologically functional antibody fragments so long as they bind, e.g.,specifically bind, to CDH6. WO 2018/185618 provides and is incorporatedherein by reference for exemplary CDH6-binding sequences, includingexemplary anti-CDH6 antibody sequences. In some embodiments, theanti-CDH6 antibody used in the ADCs disclosed herein is an internalizingantibody or internalizing antibody fragment.

The term “carcinoembryonic antigen-related cell adhesion molecule 5” or“CEACAM5,” as used herein, refers to any native form of human CEACAM5.The term encompasses full-length CEACAM5 (e.g., UniProt ReferenceSequence: P06731; SEQ ID NO:46), as well as any form of human CEACAM5that may result from cellular processing. The term also encompassesfunctional variants or fragments of human CEACAM5, including but notlimited to splice variants, allelic variants, and isoforms that retainone or more biologic functions of human CEACAM5 (i.e., variants andfragments are encompassed unless the context indicates that the term isused to refer to the wild-type protein only). CEACAM5 can be isolatedfrom a human, or may be produced recombinantly or by synthetic methods.

The term “anti-CEACAM5 antibody” or “antibody that binds to CEACAM5”refers to any form of antibody or fragment thereof that binds, e.g.,specifically binds, to CEACAM5, and encompasses monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,and biologically functional antibody fragments so long as they bind,e.g., specifically bind, to CEACAM5. US 2015/0125386 provides and isincorporated herein by reference for exemplary CEACAM5-bindingsequences, including exemplary anti-CEACAM5 antibody sequences. In someembodiments, the anti-CEACAM5 antibody used in the ADCs disclosed hereinis an internalizing antibody or internalizing antibody fragment. hMN14is an exemplary anti-human CEACAM5 antibody.

The term “cryptic family protein 1B” or “CFC1B,” as used herein, refersto any native form of human CFC1B. The term encompasses full-lengthCFC1B (e.g., UniProt Reference Sequence: P0CG36; SEQ ID NO:47), as wellas any form of human CFC1B that may result from cellular processing. Theterm also encompasses functional variants or fragments of human CFC1B,including but not limited to splice variants, allelic variants, andisoforms that retain one or more biologic functions of human CFC1B(i.e., variants and fragments are encompassed unless the contextindicates that the term is used to refer to the wild-type protein only).CFC1B can be isolated from a human, or may be produced recombinantly orby synthetic methods.

The term “anti-CFC1B antibody” or “antibody that binds to CFC1B” refersto any form of antibody or fragment thereof that binds, e.g.,specifically binds, to CFC1B, and encompasses monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,and biologically functional antibody fragments so long as they bind,e.g., specifically bind, to CFC1B. WO 2002/088170 provides and isincorporated herein by reference for exemplary CFC1B-binding sequences,including exemplary anti-CFC1B antibody sequences. In some embodiments,the anti-CFC1B antibody used in the ADCs disclosed herein is aninternalizing antibody or internalizing antibody fragment.

The term “ectonucleotide pyrophosphatase/phosphodiesterase family member3” or “ENPP3,” as used herein, refers to any native form of human ENPP3.The term encompasses full-length ENPP3 (e.g., UniProt ReferenceSequence: 014638; SEQ ID NO:48), as well as any form of human ENPP3 thatmay result from cellular processing. The term also encompassesfunctional variants or fragments of human ENPP3, including but notlimited to splice variants, allelic variants, and isoforms that retainone or more biologic functions of human ENPP3 (i.e., variants andfragments are encompassed unless the context indicates that the term isused to refer to the wild-type protein only). ENPP3 can be isolated froma human, or may be produced recombinantly or by synthetic methods.

The term “anti-ENPP3 antibody” or “antibody that binds to ENPP3” refersto any form of antibody or fragment thereof that binds, e.g.,specifically binds, to ENPP3, and encompasses monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,and biologically functional antibody fragments so long as they bind,e.g., specifically bind, to ENPP3. Donate et al. ((2016) Clin CancerRes. 22(8):1989-99) provides and is incorporated herein by reference forexemplary ENPP3-binding sequences, including exemplary anti-ENPP3antibody sequences. In some embodiments, the anti-ENPP3 antibody used inthe ADCs disclosed herein is an internalizing antibody or internalizingantibody fragment.

The term “folate receptor alpha” or “FOLR1,” as used herein, refers toany native form of human FOLR1. The term encompasses full-length FOLR1(e.g., UniProt Reference Sequence: P15328; SEQ ID NO:49), as well as anyform of human FOLR1 that may result from cellular processing. The termalso encompasses functional variants or fragments of human FOLR1,including but not limited to splice variants, allelic variants, andisoforms that retain one or more biologic functions of human FOLR1(i.e., variants and fragments are encompassed unless the contextindicates that the term is used to refer to the wild-type protein only).FOLR1 can be isolated from a human, or may be produced recombinantly orby synthetic methods.

The term “anti-FOLR1 antibody” or “antibody that binds to FOLR1” refersto any form of antibody or fragment thereof that binds, e.g.,specifically binds, to FOLR1, and encompasses monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,and biologically functional antibody fragments so long as they bind,e.g., specifically bind, to FOLR1. WO 2005/080431 and Coney et al.((1991) Cancer Res. 51(22):6125-32) provide and are incorporated hereinby reference for exemplary FOLR1-binding sequences, including exemplaryanti-FOLR1 antibody sequences. In some embodiments, the anti-FOLR1antibody used in the ADCs disclosed herein is an internalizing antibodyor internalizing antibody fragment. Farletuzumab and MOv19 are exemplaryanti-human FOLR1 antibodies.

The term “hepatitis A virus cellular receptor 1” or “HAVCR1,” as usedherein, refers to any native form of human HAVCR1. The term encompassesfull-length HAVCR1 (e.g., UniProt Reference Sequence: Q96D42; SEQ IDNO:50), as well as any form of human HAVCR1 that may result fromcellular processing. The term also encompasses functional variants orfragments of human HAVCR1, including but not limited to splice variants,allelic variants, and isoforms that retain one or more biologicfunctions of human HAVCR1 (i.e., variants and fragments are encompassedunless the context indicates that the term is used to refer to thewild-type protein only). HAVCR1 can be isolated from a human, or may beproduced recombinantly or by synthetic methods.

The term “anti-HAVCR1 antibody” or “antibody that binds to HAVCR1”refers to any form of antibody or fragment thereof that binds, e.g.,specifically binds, to HAVCR1, and encompasses monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,and biologically functional antibody fragments so long as they bind,e.g., specifically bind, to HAVCR1. Thomas et al. ((2016) Mol CancerTher. 15(12):2946-54) provides and is incorporated herein by referencefor exemplary HAVCR1-binding sequences, including exemplary anti-HAVCR1antibody sequences. In some embodiments, the anti-HAVCR1 antibody usedin the ADCs disclosed herein is an internalizing antibody orinternalizing antibody fragment.

The term “mast/stem cell growth factor receptor Kit” or “KIT,” as usedherein, refers to any native form of human KIT. The term encompassesfull-length KIT (e.g., UniProt Reference Sequence: P10721; SEQ IDNO:51), as well as any form of human KIT that may result from cellularprocessing. The term also encompasses functional variants or fragmentsof human KIT, including but not limited to splice variants, allelicvariants, and isoforms that retain one or more biologic functions ofhuman KIT (i.e., variants and fragments are encompassed unless thecontext indicates that the term is used to refer to the wild-typeprotein only). KIT can be isolated from a human, or may be producedrecombinantly or by synthetic methods.

The term “anti-KIT antibody” or “antibody that binds to KIT” refers toany form of antibody or fragment thereof that binds, e.g., specificallybinds, to KIT, and encompasses monoclonal antibodies (includingfull-length monoclonal antibodies), polyclonal antibodies, andbiologically functional antibody fragments so long as they bind, e.g.,specifically bind, to KIT. Shi et al. ((2016) Proc Natl Acad Sci USA113(33):E4784-93) and Abrams et al. ((2018) Clin Cancer Res.24(17):4297-308) provide and are incorporated herein by reference forexemplary KIT-binding sequences, including exemplary anti-KIT antibodysequences. In some embodiments, the anti-KIT antibody used in the ADCsdisclosed herein is an internalizing antibody or internalizing antibodyfragment.

The term “hepatocyte growth factor receptor” or “MET,” as used herein,refers to any native form of human MET. The term encompasses full-lengthMET (e.g., UniProt Reference Sequence: P08581; SEQ ID NO:52), as well asany form of human MET that may result from cellular processing. The termalso encompasses functional variants or fragments of human MET,including but not limited to splice variants, allelic variants, andisoforms that retain one or more biologic functions of human MET (i.e.,variants and fragments are encompassed unless the context indicates thatthe term is used to refer to the wild-type protein only). MET can beisolated from a human, or may be produced recombinantly or by syntheticmethods.

The term “anti-MET antibody” or “antibody that binds to MET” refers toany form of antibody or fragment thereof that binds, e.g., specificallybinds, to MET, and encompasses monoclonal antibodies (includingfull-length monoclonal antibodies), polyclonal antibodies, andbiologically functional antibody fragments so long as they bind, e.g.,specifically bind, to MET. Yang et al. ((2019) Acta Pharmacol Sin.)provides and is incorporated herein by reference for exemplaryMET-binding sequences, including exemplary anti-MET antibody sequences.In some embodiments, the anti-MET antibody used in the ADCs disclosedherein is an internalizing antibody or internalizing antibody fragment.

The term “mucin-16” or “MUC16,” as used herein, refers to any nativeform of human MUC16. The term encompasses full-length MUC16 (e.g.,UniProt Reference Sequence: Q8WXI7; SEQ ID NO:53), as well as any formof human MUC16 that may result from cellular processing. The term alsoencompasses functional variants or fragments of human MUC16, includingbut not limited to splice variants, allelic variants, and isoforms thatretain one or more biologic functions of human MUC16 (i.e., variants andfragments are encompassed unless the context indicates that the term isused to refer to the wild-type protein only). MUC16 can be isolated froma human, or may be produced recombinantly or by synthetic methods.

The term “anti-MUC16 antibody” or “antibody that binds to MUC16” refersto any form of antibody or fragment thereof that binds, e.g.,specifically binds, to MUC16, and encompasses monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,and biologically functional antibody fragments so long as they bind,e.g., specifically bind, to MUC16. Liu et al. ((2016) Ann Oncol.27(11):2124-30) provides and is incorporated herein by reference forexemplary MUC16-binding sequences, including exemplary anti-MUC16antibody sequences. In some embodiments, the anti-MUC16 antibody used inthe ADCs disclosed herein is an internalizing antibody or internalizingantibody fragment.

The term “zinc transporter ZIP6” or “SLC39A6,” as used herein, refers toany native form of human SLC39A6. The term encompasses full-lengthSLC39A6 (e.g., UniProt Reference Sequence:Q13433; SEQ ID NO:54), as wellas any form of human SLC39A6 that may result from cellular processing.The term also encompasses functional variants or fragments of humanSLC39A6, including but not limited to splice variants, allelic variants,and isoforms that retain one or more biologic functions of human SLC39A6(i.e., variants and fragments are encompassed unless the contextindicates that the term is used to refer to the wild-type protein only).SLC39A6 can be isolated from a human, or may be produced recombinantlyor by synthetic methods.

The term “anti-SLC39A6 antibody” or “antibody that binds to SLC39A6”refers to any form of antibody or fragment thereof that binds, e.g.,specifically binds, to SLC39A6, and encompasses monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,and biologically functional antibody fragments so long as they bind,e.g., specifically bind, to SLC39A6. Sussman et al. ((2014) Mol CancerTher. 13(12):2991-3000) provides and is incorporated herein by referencefor exemplary SLC39A6-binding sequences, including exemplaryanti-SLC39A6 antibody sequences. In some embodiments, the anti-SLC39A6antibody used in the ADCs disclosed herein is an internalizing antibodyor internalizing antibody fragment.

The term “choline transporter-like protein 4” or “SLC44A4,” as usedherein, refers to any native form of human SLC44A4. The term encompassesfull-length SLC44A4 (e.g., UniProt Reference Sequence: Q53GD3; SEQ IDNO:55), as well as any form of human SLC44A4 that may result fromcellular processing. The term also encompasses functional variants orfragments of human SLC44A4, including but not limited to splicevariants, allelic variants, and isoforms that retain one or morebiologic functions of human SLC44A4 (i.e., variants and fragments areencompassed unless the context indicates that the term is used to referto the wild-type protein only). SLC44A4 can be isolated from a human, ormay be produced recombinantly or by synthetic methods.

The term “anti-SLC44A4 antibody” or “antibody that binds to SLC44A4”refers to any form of antibody or fragment thereof that binds, e.g.,specifically binds, to SLC44A4, and encompasses monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,and biologically functional antibody fragments so long as they bind,e.g., specifically bind, to SLC44A4. Mattie et al. ((2016) Mol CancerTher. 15(11):2679-87) provides and is incorporated herein by referencefor exemplary SLC44A4-binding sequences, including exemplaryanti-SLC44A4 antibody sequences. In some embodiments, the anti-SLC44A4antibody used in the ADCs disclosed herein is an internalizing antibodyor internalizing antibody fragment.

The term “metalloreductase STEAP1” or “STEAP1,” as used herein, refersto any native form of human STEAP1. The term encompasses full-lengthSTEAP1 (e.g., UniProt Reference Sequence: Q9UHE8; SEQ ID NO:56), as wellas any form of human STEAP1 that may result from cellular processing.The term also encompasses functional variants or fragments of humanSTEAP1, including but not limited to splice variants, allelic variants,and isoforms that retain one or more biologic functions of human STEAP1(i.e., variants and fragments are encompassed unless the contextindicates that the term is used to refer to the wild-type protein only).STEAP1 can be isolated from a human, or may be produced recombinantly orby synthetic methods.

The term “anti-STEAP1 antibody” or “antibody that binds to STEAP1”refers to any form of antibody or fragment thereof that binds, e.g.,specifically binds, to STEAP1, and encompasses monoclonal antibodies(including full-length monoclonal antibodies), polyclonal antibodies,and biologically functional antibody fragments so long as they bind,e.g., specifically bind, to STEAP1. WO 2008/052187 provides and isincorporated herein by reference for exemplary STEAP1-binding sequences,including exemplary anti-STEAP1 antibody sequences. In some embodiments,the anti-STEAP1 antibody used in the ADCs disclosed herein is aninternalizing antibody or internalizing antibody fragment.

As used herein, the term “specific,” “specifically binds,” and “bindsspecifically” refers to a binding reaction between an antibody orantigen binding fragment (e.g., an anti-HER2 antibody) and a targetantigen (e.g., HER2) in a heterogeneous population of proteins and otherbiologics. Antibodies can be tested for specificity of binding bycomparing binding to an appropriate antigen to binding to an irrelevantantigen or antigen mixture under a given set of conditions. If theantibody binds to the appropriate antigen with at least 2, 5, 7, andpreferably 10 or more times more affinity than to the irrelevant antigenor antigen mixture, then it is considered to be specific. A “specificantibody” or a “target-specific antibody” is one that only binds thetarget antigen (e.g., HER2), but does not bind (or exhibits minimalbinding) to other antigens. In certain embodiments, an antibody orantigen binding fragment that specifically binds a target antigen (e.g.,HER2) has a K_(D) of less than 1×10⁻⁶ M, less than 1×10⁻⁷ M, less than1×10⁻⁸ M, less than 1×10⁻⁹ M, less than 1×10⁻¹⁰ M, less than 1×10⁻¹¹ M,less than 1×10⁻¹² M, or less than 1×10⁻¹³ M. In certain embodiments, theK_(D) is 1 μM to 500 pM. In some embodiments, the K_(D) is between 500pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

The term “epitope” refers to the portion of an antigen capable of beingrecognized and specifically bound by an antibody. When the antigen is apolypeptide, epitopes can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of thepolypeptide. The epitope bound by an antibody may be identified usingany epitope mapping technique known in the art, including X-raycrystallography for epitope identification by direct visualization ofthe antigen-antibody complex, as well as monitoring the binding of theantibody to fragments or mutated variations of the antigen, ormonitoring solvent accessibility of different parts of the antibody andthe antigen. Exemplary strategies used to map antibody epitopes include,but are not limited to, array-based oligo-peptide scanning, limitedproteolysis, site-directed mutagenesis, high-throughput mutagenesismapping, hydrogen-deuterium exchange, and mass spectrometry (see, e.g.,Gershoni et al. (2007) 21:145-56; and Hager-Braun and Tomer (2005)Expert Rev Proteomics 2:745-56).

Competitive binding and epitope binning can also be used to determineantibodies sharing identical or overlapping epitopes. Competitivebinding can be evaluated using a cross-blocking assay, such as the assaydescribed in “Antibodies, A Laboratory Manual,” Cold Spring HarborLaboratory, Harlow and Lane (1^(st) edition 1988, 2^(nd) edition 2014).In some embodiments, competitive binding is identified when a testantibody or binding protein reduces binding of a reference antibody orbinding protein to a target antigen such as HER2 (e.g., a bindingprotein comprising CDRs and/or variable domains selected from thoseidentified in Tables 2-4), by at least about 50% in the cross-blockingassay (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or anypercentage in between), and/or vice versa. In some embodiments,competitive binding can be due to shared or similar (e.g., partiallyoverlapping) epitopes, or due to steric hindrance where antibodies orbinding proteins bind at nearby epitopes (see, e.g., Tzartos, Methods inMolecular Biology (Morris, ed. (1998) vol. 66, pp. 55-66)). In someembodiments, competitive binding can be used to sort groups of bindingproteins that share similar epitopes. For example, binding proteins thatcompete for binding can be “binned” as a group of binding proteins thathave overlapping or nearby epitopes, while those that do not compete areplaced in a separate group of binding proteins that do not haveoverlapping or nearby epitopes.

The term “k_(on)” or “k_(a)” refers to the on-rate constant forassociation of an antibody to the antigen to form the antibody/antigencomplex. The rate can be determined using standard assays, such as asurface plasmon resonance, biolayer inferometry, or ELISA assay.

The term “k_(off)” or “k_(d)” refers to the off-rate constant fordissociation of an antibody from the antibody/antigen complex. The ratecan be determined using standard assays, such as a surface plasmonresonance, biolayer inferometry, or ELISA assay.

The term “K_(D)” refers to the equilibrium dissociation constant of aparticular antibody-antigen interaction. K_(D) is calculated byk_(a)/k_(d). The rate can be determined using standard assays, such as asurface plasmon resonance, biolayer inferometry, or ELISA assay.

The term “p” or “drug loading” or “drug:antibody ratio” or“drug-to-antibody ratio” or “DAR” refers to the number of drug moietiesper antibody or antigen binding fragment, i.e., drug loading, or thenumber of -L-D moieties per antibody or antigen binding fragment (Ab) inADCs of Formula (I). In ADCs comprising a splicing modulator drugmoiety, “p” refers to the number of splicing modulator compounds linkedto the antibody or antigen binding fragment. For example, if twosplicing modulator compounds (e.g., two compounds each having thestructure of D1) are linked to an antibody or antigen binding fragment,p=2. In compositions comprising multiple copies of ADCs of Formula (I),“average p” refers to the average number of -L-D moieties per antibodyor antigen binding fragment, also referred to as “average drug loading.”

A “linker” or “linker moiety” is used herein to refer to any chemicalmoiety that is capable of covalently joining a compound, usually a drugmoiety such as a splicing modulator drug moiety, to another moiety suchas an antibody or antigen binding fragment. Linkers can be susceptibleto or substantially resistant to acid-induced cleavage,peptidase-induced cleavage, light-based cleavage, esterase-inducedcleavage, and/or disulfide bond cleavage, at conditions under which thecompound or the antibody remains active.

The term “agent” is used herein to refer to a chemical compound, amixture of chemical compounds, a biological macromolecule, or an extractmade from biological materials. The term “therapeutic agent” or “drug”refers to an agent that is capable of modulating a biological processand/or has biological activity. The splicing modulator compoundsdescribed herein are exemplary therapeutic agents.

The term “chemotherapeutic agent” or “anti-cancer agent” is used hereinto refer to all agents that are effective in treating cancer regardlessof mechanism of action. Inhibition of metastasis or angiogenesis isfrequently a property of a chemotherapeutic agent. Chemotherapeuticagents include antibodies, biological molecules, and small molecules,and encompass the splicing modulator compounds described herein. Achemotherapeutic agent may be a cytotoxic or cytostatic agent. The term“cytostatic agent” refers to an agent that inhibits or suppresses cellgrowth and/or multiplication of cells. The term “cytotoxic agent” refersto a substance that causes cell death primarily by interfering with acell's expression activity and/or functioning.

As used herein, the terms “splicing modulator,” “spliceosome modulator,”or “splice modulator” refer to compounds that have anti-cancer activityby interacting with components of the spliceosome. In some embodiments,a splicing modulator alters the rate or form of splicing in a targetcell. Splicing modulators that function as inhibitory agents, forexample, are capable of decreasing uncontrolled cellular proliferation.In some embodiments, the splicing modulators may act by binding to theSF3b spliceosome complex. Such modulators may be natural compounds orsynthetic compounds. Non-limiting examples of splicing modulators andcategories of such modulators include pladienolide (e.g., pladienolide Dor pladienolide B), pladienolide derivatives (e.g., pladienolide D orpladienolide B derivatives), herboxidiene, herboxidiene derivatives,spliceostatin, spliceostatin derivatives, sudemycin, or sudemycinderivatives. As used herein, the terms “derivative” and “analog” whenreferring to a splicing modulator, or the like, means any such compoundthat retains essentially the same, similar, or enhanced biologicalfunction or activity as the original compound but has an alteredchemical or biological structure. In some embodiments, the splicingmodulator is a pladienolide or pladienolide derivative.

As used herein, a “pladienolide derivative” refers to a compound whichis structurally related to a member of the family of natural productsknown as the pladienolides and which retains one or more biologicalfunctions of the starting compound. Pladienolides were first identifiedin the bacteria Streptomyces platensis (Mizui et al. (2004) J Antibiot.57:188-96) as being potently cytotoxic and resulting in cell cyclearrest in the G1 and G2/M phases of the cell cycle (e.g., Bonnal et al.(2012) Nat Rev Drug Dis 11:847-59). There are seven naturally occurringpladienolides, pladienolide A-G (Mizui et al. (2004) J Antibiot.57:188-96; Sakai et al. (2004) J Antibiotics 57:180-7). U.S. Pat. Nos.7,884,128 and 7,816,401 describe exemplary methods of synthesizingpladienolide B and D and are each incorporated herein by reference forsuch methods. Synthesis of pladienolide B and D may also be performedusing the exemplary methods described in Kanada et al. ((2007) AngewChem Int Ed. 46:4350-5). Kanada et al. and Intl. Pub. No. WO 2003/099813describe exemplary methods for synthesizing E7107 (D11) (Compound 45 ofWO 2003/099813) from Pladienolide D (11107D of WO 2003/099813). Acorresponding U.S. Pat. No. is U.S. Pat. No. 7,550,503 to Kotake et al.Each of these references is incorporated herein for the describedsynthesis methods.

As used herein, a “splicing modulator drug moiety” refers to thecomponent of an ADC or composition that provides the structure of asplicing modulator compound, e.g., the splicing modulator (D) componentin an ADC of Formula (I), or in a composition comprising -L-D.

As used herein, a “spliceosome” refers to a ribonucleoprotein complexthat removes introns from one or more RNA segments, such as pre-mRNAsegments.

The term “homolog” refers to a molecule which exhibits homology toanother molecule, by for example, having sequences of chemical residuesthat are the same or similar at corresponding positions.

The term “inhibit” or “inhibition of,” as used herein, means to reduceby a measurable amount, and can include but does not require completeprevention or inhibition.

The term “target-negative,” “target antigen-negative,” or“antigen-negative” refers to the absence of target antigen expression bya cell or tissue. The term “target-positive,” “target antigen-positive,”or “antigen-positive” refers to the presence of target antigenexpression. For example, a cell or a cell line that does not express atarget antigen may be described as target-negative, whereas a cell orcell line that expresses a target antigen may be described astarget-positive.

The term “bystander killing” or “bystander effect” refers to the killingof target-negative cells in the presence of target-positive cells,wherein killing of target-negative cells is not observed in the absenceof target-positive cells. Cell-to-cell contact, or at least proximitybetween target-positive and target-negative cells, enables bystanderkilling. This type of killing is distinguishable from “off-targetkilling,” which refers to the indiscriminate killing of target-negativecells. “Off-target killing” may be observed in the absence oftarget-positive cells.

The terms “neoplastic disorder” and “cancer” are used hereininterchangeably to refer to the presence of cells possessingcharacteristics typical of cancer-causing cells, such as uncontrolledproliferation, immortality, metastatic potential, rapid growth andproliferation rate, and/or certain morphological features. Often, cancercells can be in the form of a tumor or mass, but such cells may existalone within a subject, or may circulate in the blood stream asindependent cells, such as leukemic or lymphoma cells. The terms“neoplastic disorder” and “cancer” includes all types of cancers andcancer metastases, including hematological malignancy, solid tumors,sarcomas, carcinomas and other solid and non-solid tumor cancers.Hematological malignancies may include B-cell malignancies, cancers ofthe blood (leukemias), cancers of plasma cells (myelomas, e.g., multiplemyeloma), or cancers of the lymph nodes (lymphomas). Exemplary B-cellmalignancies include chronic lymphocytic leukemia (CLL), follicularlymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma.Leukemias may include acute lymphoblastic leukemia (ALL), acutemyelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronicmyelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML),acute monocytic leukemia (AMoL), etc.

Lymphomas may include Hodgkin's lymphoma and non-Hodgkin's lymphoma.Other hematologic malignancies may include myelodysplasia syndrome(MDS). Solid tumors may include carcinomas such as adenocarcinoma, e.g.,breast cancer, pancreatic cancer, prostate cancer, colon or colorectalcancer, lung cancer, gastric cancer, cervical cancer, endometrialcancer, ovarian cancer, cholangiocarcinoma, glioma, melanoma, etc.

The terms “tumor” and “neoplasm” refer to any mass of tissue thatresults from excessive cell growth or proliferation, either benign ormalignant, including precancerous lesions.

The terms “tumor cell” and “neoplastic cell” are used interchangeablyand refer to individual cells or the total population of cells derivedfrom a tumor or neoplasm, including both non-tumorigenic cells andcancer stem cells. As used herein, the term “tumor cell” will bemodified by the term “non-tumorigenic” when referring solely to thosetumor cells lacking the capacity to renew and differentiate todistinguish those tumor cells from cancer stem cells.

The terms “subject” and “patient” are used interchangeably herein torefer to any animal, such as any mammal, including but not limited to,humans, non-human primates, rodents, and the like. In some embodiments,the mammal is a mouse. In some embodiments, the mammal is a human. Insome embodiments, the subject is a mouse. In some embodiments, thesubject is a human.

The term “co-administration” or administration “in combination with” oneor more therapeutic agents includes concurrent administration andconsecutive administration in any order.

A “pharmaceutical composition” refers to a preparation which is in suchform as to permit administration and subsequently provide the intendedbiological activity of the active ingredient(s) and/or to achieve atherapeutic effect, and which contains no additional components whichare unacceptably toxic to a subject to which the formulation would beadministered. The pharmaceutical composition may be sterile.

A “pharmaceutical excipient” comprises a material such as an adjuvant, acarrier, pH-adjusting and buffering agents, tonicity adjusting agents,wetting agents, preservative, and the like.

“Pharmaceutically acceptable” means approved or approvable by aregulatory agency of the Federal or a state government, or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia, for use inanimals, and more particularly in humans

A “pharmaceutically acceptable salt” is a salt that retains the desiredbiological activity of the parent compound and does not impart undesiredtoxicological effects. Examples of such salts are: (a) acid additionsalts formed with inorganic acids, for example, hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; and salts formed with organic acids, for example, acetic acid,oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid,gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid,tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (b)salts formed from elemental anions such as chlorine, bromine, andiodine. See, e.g., Haynes et al. “Commentary: Occurrence ofPharmaceutically Acceptable Anions and Cations in the CambridgeStructural Database,” J Pharmaceutical Sciences, vol. 94, no. 10 (2005),and Berge et al. “Pharmaceutical Salts,” J Pharmaceutical Sciences, vol.66, no. 1 (1977), which are incorporated by reference herein.

The term “effective amount,” as used herein, refers to the amount of acompound, ADC, or composition described herein (e.g., a splicingmodulator or an ADC) that is sufficient to perform a specifically statedpurpose, for example to produce a therapeutic effect afteradministration, such as a reduction in tumor growth rate or tumorvolume, a reduction in a symptom of cancer, or some other indicia oftreatment efficacy. An effective amount can be determined in a routinemanner in relation to the stated purpose. The term “therapeuticallyeffective amount” refers to an amount of a compound, an ADC, orcomposition described herein effective for detectable killing,reduction, and/or inhibition of the growth or spread of tumor cells, thesize or number of tumors, and/or other measure of the level, stage,progression and/or severity of the cancer. The therapeutically effectiveamount can vary depending upon the intended application (in vitro or invivo), or the subject and disease condition being treated, e.g., theweight and age of the subject, the severity of the disease condition,the manner of administration and the like, which can readily bedetermined by one of ordinary skill in the art. The term also applies toa dose that will induce a particular response in target cells, e.g.,inhibition of cell growth. The specific dose may vary depending on, forexample, the particular pharmaceutical composition, the subject andtheir age and existing health conditions or risk for health conditions,the dosing regimen to be followed, the severity of the disease, whetherit is administered in combination with other agents, timing ofadministration, the tissue to which it is administered, and the physicaldelivery system in which it is carried. In the case of cancer, atherapeutically effective amount of ADC can reduce the number of cancercells, reduce tumor size, inhibit (e.g., slow or stop) tumor metastasis,inhibit (e.g., slow or stop) tumor growth, and/or relieve one or moresymptoms.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

As used herein, “to treat” or “therapeutic” and grammatically relatedterms, refer to any improvement of any consequence of disease, such asprolonged survival, less morbidity, and/or a lessening of side effectswhich result from an alternative therapeutic modality. As is readilyappreciated in the art, full eradication of disease is encompassed butnot required for a treatment act. “Treatment” or “treat,” as usedherein, refers to the administration of a described ADC or compositionto a subject, e.g., a patient. The treatment can be to cure, heal,alleviate, relieve, alter, remedy, ameliorate, palliate, improve oraffect the disorder, the symptoms of the disorder or the predispositiontoward the disorder, e.g., a cancer. In some embodiments, in addition totreating a subject with a condition, a composition disclosed herein canalso be provided prophylactically to prevent or reduce the likelihood ofdeveloping that condition.

In some embodiments, a labeled ADC is used. Suitable “labels” includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmoieties, chemiluminescent moieties, magnetic particles, and the like.

By “protein,” as used herein, is meant at least two covalently attachedamino acids. The term encompasses polypeptides, oligopeptides, andpeptides. In some embodiments, the two or more covalently attached aminoacids are attached by a peptide bond. The protein may be made up ofnaturally occurring amino acids and peptide bonds, for example when theprotein is made recombinantly using expression systems and host cells.Alternatively, the protein may include synthetic amino acids (e.g.,homophenylalanine, citrulline, ornithine, and norleucine), orpeptidomimetic structures, i.e., “peptide or protein analogs,” such aspeptoids. Peptoids are an exemplary class of peptidomimetics whose sidechains are appended to the nitrogen atom of the peptide backbone, ratherthan to the α-carbons (as they are in amino acids), and have differenthydrogen bonding and conformational characteristics in comparison topeptides (see, e.g., Simon et al. (1992) Proc Natl Acad Sci. USA89:9367). As such, peptoids can be resistant to proteolysis or otherphysiological or storage conditions, and effective at permeating cellmembranes. Such synthetic amino acids may be incorporated in particularwhen the antibody is synthesized in vitro by conventional methods wellknown in the art. In addition, any combination of peptidomimetic,synthetic and naturally occurring residues/structures can be used.“Amino acid” also includes imino acid residues, such as proline andhydroxyproline. The amino acid “R group” or “side chain” may be ineither the (L)- or the (S)-configuration. In a specific embodiment, theamino acids are in the (L)- or (S)-configuration.

A “recombinant protein” is a protein made using recombinant techniquesusing any techniques and methods known in the art, i.e., through theexpression of a recombinant nucleic acid. Methods and techniques for theproduction of recombinant proteins are well known in the art.

An “isolated” protein is unaccompanied by at least some of the materialwith which it is normally associated in its natural state, for exampleconstituting at least about 5%, or at least about 50% by weight of thetotal protein in a given sample. It is understood that the isolatedprotein may constitute from 5% to 99.9% by weight of the total proteincontent depending on the circumstances. For example, the protein may bemade at a significantly higher concentration through the use of aninducible promoter or high expression promoter, such that the protein ismade at increased concentration levels. The definition includes theproduction of an antibody in a wide variety of organisms and/or hostcells that are known in the art.

For amino acid sequences, sequence identity and/or similarity may bedetermined using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith andWaterman (1981) Adv Appl Math. 2:482, the sequence identity alignmentalgorithm of Needleman and Wunsch (1970) J Mol Biol. 48:443, the searchfor similarity method of Pearson and Lipman (1988) Proc Nat Acad Sci.USA 85:2444, computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fitsequence program described by Devereux et al. (1984) Nucl Acid Res.12:387-95, preferably using the default settings, or by inspection.Preferably, percent identity is calculated by FastDB based upon thefollowing parameters: mismatch penalty of 1; gap penalty of 1; gap sizepenalty of 0.33; and joining penalty of 30 (“Current Methods in SequenceComparison and Analysis,” Macromolecule Sequencing and Synthesis,Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss,Inc).

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle (1987) J MolEvol. 35:351-60; the method is similar to that described by Higgins andSharp (1989) CABIOS 5:151-3. Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin: Altschul et al. (1990) J Mol Biol. 215:403-10; Altschul et al.(1997) Nucl Acid Res. 25:3389-402; and Karin et al. (1993) Proc NatlAcad Sci. USA 90:5873-87. A particularly useful BLAST program is theWU-BLAST-2 program which was obtained from Altschul et al. (1996)Methods in Enzymology 266:460-80. WU-BLAST-2 uses several searchparameters, most of which are set to the default values. The adjustableparameters are set with the following values: overlap span=I, overlapfraction=0.125, word threshold (T)=II. The HSP S and HSP S2 parametersare dynamic values and are established by the program itself dependingupon the composition of the particular sequence and composition of theparticular database against which the sequence of interest is beingsearched; however, the values may be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al. (1997) Nucl Acid Res. 25:3389-402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions, charges gap lengths of k a cost of 10+k;Xu set to 16, and Xg set to 40 for database search stage and to 67 forthe output stage of the algorithms. Gapped alignments are triggered by ascore corresponding to about 22 bits.

Generally, the amino acid homology, similarity, or identity betweenproteins disclosed herein and variants thereof, including variants oftarget antigens (such as HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5,CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, orSTEAP1) and variants of antibody variable domains (including individualvariant CDRs), are at least 80% to the sequences depicted herein, e.g.,homologies or identities of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, almost 100%, or 100%.

In a similar manner, “percent (%) nucleic acid sequence identity” withrespect to the nucleic acid sequence of the antibodies and otherproteins identified herein is defined as the percentage of nucleotideresidues in a candidate sequence that are identical with the nucleotideresidues in the coding sequence of the antigen binding protein. Aspecific method utilizes the BLASTN module of WU-BLAST-2 set to thedefault parameters, with overlap span and overlap fraction set to 1 and0.125, respectively.

While the site or region for introducing an amino acid sequencevariation is predetermined, the mutation per se need not bepredetermined. For example, in order to optimize the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed antigen binding protein CDRvariants screened for the optimal combination of desired activity.Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known, for example, MI3 primermutagenesis and PCR mutagenesis

“Alkyl” or “alkyl group,” as used herein, means a straight-chain,branched, or cyclic hydrocarbon chain that is completely saturated. Incertain embodiments, alkyl groups may contain 1-8 carbon atoms(“C₁-C₈alkyl”). In certain embodiments, alkyl groups may contain 1-6carbon atoms (“C₁-C₆alkyl”). In certain embodiments, alkyl groupscontain 1-3 carbon atoms. In still other embodiments, alkyl groupscontain 2-3 carbon atoms, and in yet other embodiments alkyl groupscontain 1-2 carbon atoms.

“Alkylalkoxy,” as used herein, means an alkyl group substituted with analkoxy group. “Alkoxy”, as used herein, refers to an alkyl group, aspreviously defined, attached to the principal carbon chain through anoxygen (“alkoxy”) atom.

“Alkylamino,” as used herein, means an alkyl group substituted with anamino group. “Amino,” as used herein, refers to —NH₂, —NH(alkyl), or—N(alkyl)(alkyl).

“Alkylhydroxy,” as used herein, means an alkyl group substituted with anamino group. “Hydroxy” or “hydroxyl,” as used herein, refers to —OH.

“Alkylene” refers to a divalent radical of an alkyl group. For example,—CH₂—, —CH₂CH₂—, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—, and—CH₂CH₂CH₂CH₂CH₂CH₂— refer to methylene, ethylene, n-propylene,n-butylene, n-pentylene, and n-hexylene, respectively.

“Carbocycle,” as used herein, includes both aromatic (e.g., aryl) andnon-aromatic (e.g., cycloalkyl) groups. In certain embodiments,carbocycle groups contain 3-10 carbon atoms (“3 to 10 memberedcarbocycle”). In certain embodiments, carbocycle groups contain 3-8carbon atoms (“3 to 8 membered carbocycle”). In certain embodiments,carbocycle groups contain 3-6 carbon atoms (“3 to 6 memberedcarbocycle”). In certain embodiments, carbocycle groups contain 3-5carbon atoms (“3 to 5 membered carbocycle”).

“Halogen” refers to a radical of any halogen, e.g., —F, —Cl, —Br, or —I.

The terms “heterocycle”, “heterocyclyl”, and “heterocyclic” as usedherein, mean a monocyclic heterocycle, a bicyclic heterocycle, or atricyclic heterocycle containing at least one heteroatom in the ring.

The monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7, or 8-membered ringcontaining at least one heteroatom independently chosen from O, N, andS. In some embodiments, the heterocycle is a 3- or 4-membered ringcontaining one heteroatom chosen from O, N and S. In some embodiments,the heterocycle is a 5-membered ring containing zero or one double bondand one, two or three heteroatoms chosen from O, N and S. In someembodiments, the heterocycle is a 6-, 7-, or 8-membered ring containingzero, one or two double bonds and one, two or three heteroatoms chosenfrom O, N and S. Representative examples of monocyclic heterocycleinclude, but are not limited to, azetidinyl, azepanyl, aziridinyl,diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, dihydropyranyl (including3,4-dihydro-2H-pyran-6-yl), 1,3-dithiolanyl, 1,3-dithianyl,imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl,isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl,oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl,pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl,tetrahydrofuranyl, tetrahydropyranyl (includingtetrahydro-2H-pyran-4-yl), tetrahydrothienyl, thiadiazolinyl,thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl,1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, andtrithianyl.

The bicyclic heterocycles of the present disclosure may include amonocyclic heterocycle fused to an aryl group, or a monocyclicheterocycle fused to a monocyclic cycloalkyl, or a monocyclicheterocycle fused to a monocyclic cycloalkenyl, or a monocyclicheterocycle fused to a monocyclic heterocycle having a total of 5 to 12ring atoms. Examples of bicyclic heterocycles include, but are notlimited to, 3,4-dihydro-2H-pyranyl, 1,3-benzodioxolyl,1,3-benzodithiolyl, 2,3-dihydro-1,4-benzodioxinyl,2,3-dihydro-1-benzofuranyl, 2,3-dihydro-1-benzothienyl,2,3-dihydro-1H-indolyl, and 1,2,3,4-tetrahydroquinolinyl.

The terms “heterocycle”, “heterocyclyl”, and “heterocyclic” encompassheteroaryls. “Heteroaryl” refers to a cyclic moiety having one or moreclosed rings, with one or more heteroatoms (oxygen, nitrogen or sulfur)in at least one of the rings, wherein at least one of the rings isaromatic, and wherein the ring or rings may independently be fused,and/or bridged. Examples include without limitation phenyl, thiophenyl,triazolyl, pyridinyl, pyrimidinyl, pyridazinyl, and pyrazinyl.

As described herein, compounds of the disclosure may contain “optionallysubstituted” moieties. In general, the term “substituted,” whetherpreceded by the term “optionally” or not, means that one or morehydrogens of the designated moiety are replaced with a suitablesubstituent. Unless otherwise indicated, an “optionally substituted”group may have a suitable substituent at each substitutable position ofthe group, and when more than one position in any given structure may besubstituted with more than one substituent chosen from a specifiedgroup, the substituent may be either the same or different at eachposition. Combinations of substituents envisioned under this disclosureare preferably those that result in the formation of stable orchemically feasible compounds.

One skilled in the art will be understand that “substitution” or“substituted with” or “absent” includes the implicit proviso that suchsubstitution or absence is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution orabsence results in a stable compound, e.g., which does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc. For purposes of this disclosure, the heteroatoms suchas nitrogen may have hydrogen substituents, and/or any permissiblesubstituents of organic compounds described herein which satisfy thevalences of the heteroatoms.

“Stable” refers to compounds that are not substantially alteredchemically and/or physically when subjected to conditions to allow fortheir production, detection, and, in certain embodiments, theirrecovery, purification, and use for one or more of the purposesdisclosed herein. In some embodiments, a stable compound or chemicallyfeasible compound is one that is not substantially altered when kept ata temperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week. In someembodiments, the compounds disclosed herein are stable.

Enantiomers taught herein may include “enantiomerically pure” isomersthat comprise substantially a single enantiomer, for example, greaterthan or equal to 90%, 92%, 95%, 98%, or 99%, or equal to 100% of asingle enantiomer, at a particular asymmetric center or centers. An“asymmetric center” or “chiral center” refers to a tetrahedral carbonatom that comprises four different substituents.

The compounds described herein may also contain unnatural proportions ofatomic isotopes at one or more of the atoms that constitute suchcompounds. For example, the compounds may be radiolabeled withradioactive isotopes, such as, for example, deuterium (²H), tritium(³H), carbon-13 (¹³C), or carbon-14 (¹⁴C). All isotopic variations ofthe compounds disclosed herein, whether radioactive or not, are intendedto be encompassed within the scope of the present disclosure. Inaddition, all tautomeric forms of the compounds described herein areintended to be within the scope of the claimed disclosure.

Antibody-Drug Conjugates

The antibody-drug conjugate (ADC) compounds of the present disclosureinclude those with anti-cancer activity. In particular, the ADCcompounds include an antibody or antigen binding fragment (including anantigen binding fragment thereof) conjugated (i.e., covalently attachedby a linker) to a drug moiety (e.g., a splicing modulator), wherein thedrug moiety when not conjugated to an antibody or antigen bindingfragment has a cytotoxic or cytostatic effect. In various embodiments,the drug moiety when not conjugated to an antibody or antigen bindingfragment is capable of binding to and/or interacting with the SF3bspliceosome complex. In various embodiments, the drug moiety when notconjugated to an antibody or antigen binding fragment is capable ofmodulating in vitro and/or in vivo RNA splicing. By targeting RNAsplicing, in various embodiments, the drug moieties and ADCs disclosedherein are potent antiproliferative agents. In various embodiments, thedrug moieties and ADCs disclosed herein can target both activelydividing and quiescent cells.

In various embodiments, the present disclosure is based, at least inpart, on the discovery that certain biologically active splicingmodulators may provide improved properties when used in ADCs. While asplicing modulator may show desirably improved features (e.g., robustSF3b spliceosome complex binding, potent modulation of RNA splicing)when used on its own, in various embodiments, the splicing modulator mayexhibit fewer of the same desirably improved features when conjugated toan antibody or antigen binding fragment. Thus, the development andproduction of an ADC for use as a human therapeutic agent, e.g., as anoncologic agent, may require more than the identification of an antibodycapable of binding to a desired target or targets and attaching to adrug used on its own to treat cancer. Linking the antibody to the drugmay have significant effects on the activity of one or both of theantibody and the drug, effects which will vary depending on the type oflinker and/or drug chosen. In some embodiments, therefore, thecomponents of the ADC are selected to (i) retain one or more therapeuticproperties exhibited by the antibody and drug moieties in isolation,(ii) maintain the specific binding properties of the antibody or antigenbinding fragment; (iii) optimize drug loading and drug-to-antibodyratios; (iv) allow delivery, e.g., intracellular delivery, of the drugmoiety via stable attachment to the antibody or antigen bindingfragment; (v) retain ADC stability as an intact conjugate untiltransport or delivery to a target site; (vi) minimize aggregation of theADC prior to or after administration; (vii) allow for the therapeuticeffect, e.g., cytotoxic effect, of the drug moiety after cleavage orother release mechanism in the cellular environment; (viii) exhibit invivo anti-cancer treatment efficacy comparable to or superior to that ofthe antibody and drug moieties in isolation; (ix) minimize off-targetkilling by the drug moiety; and/or (x) exhibit desirable pharmacokineticand pharmacodynamics properties, formulatability, andtoxicologic/immunologic profiles. Each of these properties may be neededto identify an improved ADC for therapeutic use (Ab et al. (2015) MolCancer Ther. 14:1605-13).

In various embodiments, the ADCs disclosed herein exhibit unexpectedlyfavorable properties in some or each of the categories listed above. Forinstance, in some embodiments, the ADC constructs disclosed hereinexhibit surprisingly favorable drug loading, aggregation, and/orstability profiles, and/or preserve antibody binding function, drugactivity, and/or improved bystander killing, while reducing off-targetkilling, as compared to ADCs comprising an alternate linker and/or drugmoiety (e.g., an alternate splicing modulator). In some embodiments, ADCconstructs disclosed herein demonstrate superior stability, activity,potency, or other effect (measured in vivo or in vitro) as compared toADCs using an alternate linker and/or drug moiety (e.g., an alternatesplicing modulator). In some embodiments, the ADC constructs disclosedherein exhibit in vivo treatment efficacy when administered as a singledose. In some embodiments, the ADC constructs disclosed herein aresurprisingly stable as compared to ADCs using an alternate linker and/ordrug moiety (e.g., an alternate splicing modulator).

The ADC compounds of the present disclosure may selectively deliver aneffective dose of a cytotoxic or cytostatic agent to cancer cells or totumor tissue. It has been discovered that the disclosed ADCs have potentcytotoxic and/or cytostatic activity against cells expressing therespective target antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6,CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4,STEAP1). In some embodiments, the cytotoxic and/or cytostatic activityof the ADC is dependent on target antigen expression in a cell. In someembodiments, the disclosed ADCs are particularly effective at killingcancer cells expressing a target antigen while minimizing off-targetkilling. In some embodiments, the disclosed ADCs do not exhibit acytotoxic and/or cytostatic effect on cancer cells that do not express atarget antigen.

Exemplary HER2-expressing cancers include but are not limited to breastcancer, gastric cancer, bladder cancer, urothelial cell carcinoma,esophageal cancer, lung cancer (e.g., lung adenocarcinoma), uterinecancer (e.g., uterine serous endometrial carcinoma), salivary ductcarcinoma, cervical cancer, endometrial cancer, and ovarian cancer(English et al. (2013) Mol Diagn Ther. 17:85-99).

Exemplary CD138-expressing cancers include but are not limited tointrathoracic cancer (e.g., lung cancer, mesothelioma), skin cancer(e.g., basal cell carcinoma, squamous cell carcinoma), head and neckcancer (e.g., laryngeal, hypopharynx, nasopharyngeal), breast cancer,urogenital cancer (e.g., cervical cancer, ovarian cancer, endometrialcancer, prostate cancer, bladder cancer, urothelial cancer),hematological malignancies (e.g., myeloma such as multiple myeloma,B-cell malignancies, Hodgkin's lymphoma), and thyroid cancer (Szatmäriet al. (2015) Dis Markers 2015:796052).

Exemplary EPHA2-expressing cancers include breast cancer, brain cancer,ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer,lung cancer, prostate cancer, melanoma, esophageal cancer, and gastriccancer (Tandon et al. (2011) Expert Opin Ther Targets 15(1):31-51).

In some embodiments, cleavage of an ADC releases the splicing modulatorfrom the antibody or antigen binding fragment and linker. In someembodiments, the linker and/or splicing modulator is designed tofacilitate bystander killing (the killing of neighboring cells). In someembodiments, the linker and/or splicing modulator is designed tofacilitate bystander killing through cleavage after cellularinternalization and diffusion of the linker-drug moiety and/or the drugmoiety alone to neighboring cells. In some embodiments, the linkerpromotes cellular internalization. In some embodiments, the linker isdesigned to minimize cleavage in the extracellular environment andthereby reduce toxicity to off-target tissue (e.g., non-canceroustissue), while preserving ADC binding to target tissue and bystanderkilling of cancerous tissue that does not express an antigen targeted bythe antibody or antigen binding fragment of an ADC, but surrounds targetcancer tissue expressing that antigen. In some embodiments, the drugmoiety, or the catabolite of the drug moiety produced by cleavage of anADC, is designed to facilitate uptake by target cells or by neighboringcells (i.e., cell permeable). Such drug moieties and catabolites may bereferred to herein as “bystander active,” whereas drug moieties orcatabolites with reduced cell permeability may be referred to as“bystander inactive.”

In some embodiments, the disclosed ADCs also demonstrate bystanderkilling activity, but low off-target cytotoxicity. Without being boundby theory, the bystander killing activity of an ADC may be particularlybeneficial where its penetration into a solid tumor is limited and/ortarget antigen expression among tumor cells is heterogeneous. In someembodiments, an ADC comprising a cleavable linker is particularlyeffective at bystander killing and/or demonstrates improved bystanderkilling activity, relative to comparable treatment with an ADCcomprising a non-cleavable linker. In some embodiments, the ADCsdisclosed herein exhibit improved solubility and target cell penetranceover the drug moieties on their own. In some embodiments, the ADCsdisclosed herein exhibit improved cytotoxicity over that of the drugmoiety on its own. In some embodiments, ADCs disclosed herein use drugmoieties that exhibit lower cytotoxicity, when evaluated as astand-alone drug, yet are surprisingly better than ADCs comprising otherdrug moieties which have higher cytotoxicity when evaluated as astand-alone drug. In some embodiments, cleavage and release of thesplicing modulator improves cytotoxicity of the ADC, relative tocomparable treatment with an ADC comprising a non-cleavable linker. Inother embodiments, cleavage and release of the splicing modulator is notrequired for an ADC to possess a desirable biological activity. In someembodiments, an ADC comprising a non-cleavable linker having increasedspacer length (e.g., ADL12) provides the same or similar cytotoxicityrelative to comparable treatment with an ADC comprising a cleavablelinker (e.g., ADL1, ADL5) and surprisingly superior cytotoxicityrelative to comparable treatment with an ADC comprising a shorternon-cleavable linker. In some embodiments, an ADC comprising anon-cleavable linker having increased spacer length without a carbonylgroup (e.g., ADL12) provides the same or similar cytotoxicity relativeto comparable treatment with an ADC comprising a cleavable linker (e.g.,ADL1, ADL5) and surprisingly superior cytotoxicity relative tocomparable treatment with an ADC comprising a non-cleavable linkerhaving the same or similar spacer length with a carbonyl group (e.g.,ADL10). In some embodiments, the removal of a carbonyl group from anon-cleavable MC linker (e.g., ADL12) can result in a greater than50-fold, greater than 75-fold, greater than 100-fold, greater than150-fold, or greater than 200-fold increase in cytotoxicity, relative tocomparable treatment with an ADC comprising an unmodified non-cleavableMC linker (e.g., ADL10). In some embodiments, the removal of a carbonylgroup from a non-cleavable MC linker (e.g., ADL12) and increased spacerlength (e.g., the addition of at least one spacer unit) can result in agreater than 50-fold, greater than 75-fold, greater than 100-fold,greater than 150-fold, or greater than 200-fold increase incytotoxicity, relative to comparable treatment with an ADC comprising anunmodified non-cleavable MC linker (e.g., ADL10).

Provided herein are ADC compounds comprising an antibody or antigenbinding fragment thereof (Ab) which targets a tumor cell, a splicingmodulator drug moiety (D), and a linker moiety (L) that covalentlyattaches Ab to D. In certain aspects, the antibody or antigen bindingfragment is able to bind to a tumor-associated antigen (e.g., HER2,CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1,KIT, MET, MUC16, SLC39A6, SLC44A4, STEAP1) with high specificity andhigh affinity. In certain embodiments, the antibody or antigen bindingfragment is internalized into a target cell upon binding, e.g., into adegradative compartment in the cell. In various embodiments, ADCs thatinternalize upon binding to a target cell, undergo degradation, andrelease the splicing modulator drug moiety to kill cancer cells may beused. The splicing modulator drug moiety may be released from theantibody and/or the linker moiety of the ADC by enzymatic action,hydrolysis, oxidation, or any other mechanism.

An exemplary ADC has Formula (I):

Ab-(L-D)_(p)  (I)

wherein Ab=an antibody or antigen binding fragment, L=a linker moiety,D=a splicing modulator drug moiety, and p=the number of splicingmodulator drug moieties per antibody or antigen binding fragment.

In certain preferred embodiments, the drug-targeting moiety for use inthe described ADCs and compositions is an antibody or antigen bindingfragment. Other exemplary drug-targeting moieties for use in thedescribed ADCs and compositions are also provided and described herein.In some embodiments, a drug-targeting moiety can be any one of a varietyof cell-binding agents and non-antibody scaffolds. In some embodiments,the drug-targeting moiety is a cell-binding agent. As used herein, theterm “cell-binding agent” refers to any agent that is capable of bindingto an animal (e.g., human) cell and delivering a drug moiety (e.g., asplicing modulator drug moiety as disclosed herein). The termencompasses the exemplary antibodies and antigen binding fragmentsdisclosed herein (e.g., monoclonal antibodies and fragments thereof suchas Fabs and scFVs). The term further encompasses exemplary cell-bindingagents such as DARPins, duobodies, bicyclic peptides, nanobodies,centyrins, MSH (melanocyte-stimulating hormone), receptor-Fc fusionmolecules, T-cell receptor structures, steroid hormones such asandrogens and estrogens, growth factors, colony-stimulating factors suchas EGF, and other non-antibody scaffolds. In various embodiments,non-antibody scaffolds can broadly fall into two structural classes,namely domain-sized compounds (approximately 6-20 kDa) and constrainedpeptides (approximately 2-4 kDa). Exemplary domain-sized scaffoldsinclude but are not limited to affibodies, affilins, anticalins,atrimers, DARPins, FN3 scaffolds (e.g., adnectins and centyrins),fynomers, Kunitz domains, pronectins, O-bodies, and receptor-Fc fusionproteins, whereas exemplary constrained peptides include avimers,bicyclic peptides, and Cys-knots. In some embodiments, thedrug-targeting moiety used in the described ADCs and compositions isselected from an affibody, an affilin, an anticalin, an atrimer, aDARPin, a FN3 scaffold such as an adnectin or a centyrin, a fynomer, aKunitz domain, a pronectin, an O-body, an avimer, a bicyclic peptide,and a Cys-knot. In some embodiments, the drug-targeting moiety used inthe described ADCs and compositions is a receptor-Fc fusion protein,e.g., a HER2-Fc chimeric fusion protein. Non-antibody scaffolds arereviewed, e.g., in Vazquez-Lombardi et al. (2015) Drug Dis Today20(10):1271-83.

Antibodies

The antibody or antigen binding fragment (Ab) of Formula (I) includeswithin its scope any antibody or antigen binding fragment thatspecifically binds to a target antigen on a cancer cell. The antibody orantigen binding fragment may bind to a target antigen with adissociation constant (K_(D)) of mM, ≤100 nM or ≤10 nM, or any amount inbetween, as measured by, e.g., BIAcore® analysis. In certainembodiments, the K_(D) is 1 pM to 500 pM. In some embodiments, the K_(D)is between 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

In some embodiments, the antibody or antigen binding fragment is afour-chain antibody (also referred to as an immunoglobulin or afull-length or intact antibody), comprising two heavy chains and twolight chains. In some embodiments, the antibody or antigen bindingfragment is a two-chain half body (one light chain and one heavy chain),or an antigen binding fragment of an immunoglobulin. In someembodiments, the antibody or antigen binding fragment is an antigenbinding fragment of an immunoglobulin that retains the ability to bind atarget cancer antigen and/or provide a function of an immunoglobulin.

In some embodiments, the antibody or antigen binding fragment is anantibody or antigen binding fragment thereof. In some embodiments, theantibody or antigen binding fragment is an internalizing antibody orinternalizing antigen binding fragment thereof. In some embodiments, theinternalizing antibody or internalizing antigen binding fragment thereofbinds to a target cancer antigen expressed on the surface of a cell andenters the cell upon binding. In some embodiments, the splicingmodulator drug moiety of the ADC is released from the antibody orantigen binding fragment of the ADC after the ADC enters and is presentin a cell expressing the target cancer antigen (i.e., after the ADC hasbeen internalized), e.g., by cleavage, by degradation of the antibody orantigen binding fragment, or by any other suitable release mechanism.

Amino acid sequences of exemplary antibodies of the present disclosureare set forth in Tables 2-4.

TABLE 1 Antibodies mAb Type Target trastuzumab (AB185) humanizedHER2/NEU B-B4 (AB205) murine CD138 (syndecan-1) 1C1 (AB206) humanizedEPHA2

TABLE 2 Amino acid sequences of mAb variable regions mAb IgG chainSEQ ID NO Amino acid sequence trastuzumab Heavy chain 19EVQLVESGGGLVQPGGSLRLSCAASGF (AB185) NIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYL QMNSLRAEDTAVYYCSRWGGDGFYAMD YWGQGTLVTVSStrastuzumab Light chain 20 DIQMTQSPSSLSASVGDRVTITCRASQ (AB185)DVNTAVAWYQQKPGKAPKLLIYSASFL YSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKR T B-B4 Heavy chain 21QVQLQQSGSELMMPGASVKISCKATGY (AB205) TFSNYWIQRPGHGLEWIGEILPGTGRTIYNEKFKGKATFTADISSNTVQMQLSS LTSEDSAVYYCARRDYYGNFYYAMDYW GQGTSVTVSS B-B4Light chain 22 DIQMTQSTSSLSASLGDRVTISCSASQ (AB205)GINNYLNWYQQKPDGTVELLIYYTSIL QSGVPSRFSGSGSGTDYSLTISNLEPEDIGTYYCQQYSKLPRTFGGGTKLEIK 1C1 Heavy chain 23EVQLLESGGGLVQPGGSLRLSCAASGF (AB206) TFSHYMMAWVRQAPGKGLEWVSRIGPSGGPTHYADSVKGRFTISRDNSKNTLYL QMNSLRAEDTAVYYCAGYDSGYDYVAVAGPAE-YFQHWGQGTLVTVSS 1C1 Light chain 24 DIQMTQSPSSLSASVGDRVTITCRASQ(AB206) SISTWLAWYQQKPGKAPKLLIYKASNL HTGVPSRFSGSGSGTEFSLTISGLQPDDFATYYCQQYNSYS-RTFGQGTKVEIK

TABLE 3 Amino acid sequences of mAb CDRs mAb IgG chain SEQ ID NOAmino acid sequence trastuzumab HCDR1 1 GFNIKDTYIH (AB185) trastuzumabHCDR2 2 RIYPTNGYTRYADSVKG (AB185) trastuzumab HCDR3 3 WGGDGFYAMDV(AB185) trastuzumab LCDR1 4 RASQDVNTAVAW (AB185) trastuzumab LCDR2 5SASFLES (AB185) trastuzumab LCDR3 6 QQHYTTPPT (AB185) B-B4 HCDR1 7 NYWIE(AB205) B-B4 HCDR2 8 ILPGTGRTIYNEKFKGKA (AB205) B-B4 HCDR3 9RDYYGNFYYAMDY (AB205) B-B4 LCDR1 10 ASQGINNYLN (AB205) B-B4 LCDR2 11TSTLQS (AB205) B-B4 LCDR3 12 QQYSKLPRT (AB205) 1C1 HCDR1 13 HYMMA(AB206) 1C1 HCDR2 14 RIGPSGGPTHYADSVKG (AB206) 1C1 HCDR3 15YDSGYDYVAVAGPAE-YFQH (AB206) 1C1 LCDR1 16 RASWSISTWLA (AB206) 1C1 LCDR217 KASNLHT (AB206) 1C1 LCDR3 18 QQYNSYS-RT (AB206)

TABLE 4 Amino acid sequences of full-length mAb Ig chains mAb IgG chainClass SEQ ID NO Amino acid sequence trastuzumab Heavy chain IgG1 25EVQLVESGGGLVQPGGSLRLSCAA (AB185) SGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA DTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTS GVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGKtrastuzumab Light chain kappa 26 DIQMTQSPSSLSASVGDRVTITCR (AB185)ASQDVNTAVAWYQQKPGKAPKLLI YSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA KVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC B-B4 Heavy chain IgG2a27 QVQLQQSGSELMMPGASVKISCKA (AB205) TGYTFSNYWIQRPGHGLEWIGEILPGTGRTIYNEKFKGKATFTADISS NTVQMQLSSLTSEDSAVYYCARRDYYGNFYYAMDYWGQGTSVTVSSAK TTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGV HTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIV PRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKD DPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGK EFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSL TCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQ KSNWEAGNTFTCSVLHEGLHNHHT EKSLSHSPG B-B4Light chain kappa 28 DIQMTQSTSSLSASLGDRVTISCS (AB205)ASQGINNYLNWYQQKPDGTVELLI YYTSTLQSGVPSRFSGSGSGTDYSLTISNLEPEDIGTYYCQQYSKLPR TFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDI NVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLIKDEYERHNSY TCEATHKTSTSPIVKSFNRNEC 1C1 Heavy chain IgG1 29EVQLLESGGGLVQPGGSLRLSCAA (AB206) SGFTFSHYMMAWVRQAPGKGLEWVSRIGPSGGPTHYADSVKGRFTISR DNSKNTLYLQMNSLRAEDTAVYYCAGYDSGYDYVAVAGPAEYFQHWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRIPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G 1C1 Light chain kappa 30DIQMTQSPSSLSASVGDRVTITCR (AB206) ASQSISTWLAWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTEFS LTISGLQPDDFATYYCQQYNSYSRTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS KDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC

TABLE 5 Exemplary target antigen amino acid sequences Antigen SEQ ID NOAmino acid sequence HER2/NEU 31MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV CD138 32MRRAALWLWLCALALSLQPALPQIVATNLPPEDQDGSGDDSDNFSGSGAGALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAASTSTLPAGEGPKEGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTHLASTTTATTAQEPATSHPHRDMQPGHHETSTPAGPSQADLHTPHTEDGGPSATERAAEDGASSQLPAAEGSGEQDFTFETSGENTAVVAVEPDRRNQSPVDQGATGASQGLLDRKEVLGGVIAGGLVGLIFAVCLVGFMLYRMKKKDEGSYSLEEPKQANGGAYQKPTKQEEFYA EPHA2 33MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVICEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIGGVAVGVVLLLVLAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTYVDPHTYEDPNQAVLKFTTEIHPSCVTRQKVIGAGEFGEVYKGMLKTSSGKKEVPVAIKTLKAGYTEKQRVDFLGEAGIMGQFSHHNIIRLEGVISKYKPMMIITEYMENGALDKFLREKDGEFSVLQLVGMLRGIAAGMKYLANMNYVHRDLAARNILVNSNLVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWTAPEAISYRKFTSASDVWSFGIVMWEVMTYGERPYWELSNHEVMKAINDGFRLPTPMDCPSAIYQLMMQCWQQERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSGSEGVPFRTVSEWLESIKMQQYTEHFMAAGYTAIEKVVQMTNDDIKRIGVRLPGHQKRIAYSLLGLKDQVNTVGIPI MSLN 43MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVISLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA FOLH1 44MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYARTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVFELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA CDH6 45MRTYRYFLLLFWVGQPYPTLSTPLSKRTSGFPAKKRALELSGNSKNELNRSKRSWMWNQFFLLEEYTGSDYQYVGKLHSDQDRGDGSLKYILSGDGAGDLFIINENTGDIQATKRLDREEKPVYILRAQAINRRTGRPVEPESEFIIKIHDINDNEPIFTKEVYTATVPEMSDVGTFVVQVTATDADDPTYGNSAKVVYSILQGQPYFSVESETGIIKTALLNMDRENREQYQVVIQAKDMGGQMGGLSGTTIVNITLTDVNDNPPRFPQSTYQFKTPESSPPGTPIGRIKASDADVGENAEIEYSITDGEGLDMFDVITDQETQEGIITVKKLLDFEKKKVYTLKVEASNPYVEPRFLYLGPFKDSATVRIVVEDVDEPPVFSKLAYILQIREDAQINTTIGSVTAQDPDAARNPVKYSVDRHTDMDRIFNIDSGNGSIFTSKLLDRETLLWHNITVIATEINNPKQSSRVPLYIKVLDVNDNAPEFAEFYETFVCEKAKADQLIQTLHAVDKDDPYSGHQFSFSLAPEAASGSNFTIQDNKDNTAGILTRKNGYNRHEMSTYLLPVVISDNDYPVQSSTGTVTVRVCACDHHGNMQSCHAEALIHPTGLSTGALVAILLCIVILLVTVVLFAALRRQRKKEPLIISKEDIRDNIVSYNDEGGGEEDTQAFDIGTLRNPEAIEDNKLRRDIVPEALFLPRRTPTARDNTDVRDFINQRLKENDTDPTAPPYDSLATYAYEGTGSVADSLSSLESVTTDADQDYD YLSDWGPRFKKLADMYGGVDSDKDSCEACAM5 46 MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRITVITITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLILLSVIRNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRILTLFNVIRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVALI CFC1B 47MTWRHHVRLLFTVSLALQIINLGNSYQREKHNGGREEVTKVATQKHRQSPLNWTSSHFGEVTGSAEGWGPEEPLPYSWAFGEGASARPRCCRNGGTCVLGSFCVCPAHFTGRYCEHDQRRSECGALEHGAWTLRACHLCRCIFGALHCLPLQTPDRCDPKDFLASHAHGPSAGGAPSLLLLLPCALLHRLLRPDAPAHPRSLVPSVLQRERRPCGRPGLGHRL ENPP3 48MESTLTLATEQPVKKNTLKKYKIACIVLLALLVIMSLGLGLGLGLRKLEKQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCVESTRIWMCNKFRCGETRLEASLCSCSDDCLQRKDCCADYKSVCQGETSWLEENCDTAQQSQCPEGFDLPPVILFSMDGFRAEYLYTWDTLMPNINKLKTCGIHSKYMRAMYPTKTFPNHYTIVTGLYPESHGIIDNNMYDVNLNKNFSLSSKEQNNPAWWHGQPMWLTAMYQGLKAATYFWPGSEVAINGSFPSIYMPYNGSVPFEERISTLLKWLDLPKAERPRFYTMYFEEPDSSGHAGGPVSARVIKALQVVDHAFGMLMEGLKQRNLHNCVNIILLADHGMDQTYCNKMEYMTDYFPRINFFYMYEGPAPRIRAHNIPHDFFSFNSEEIVRNLSCRKPDQHFKPYLTPDLPKRLHYAKNVRIDKVHLFVDQQWLAVRSKSNTNCGGGNHGYNNEFRSMEAIFLAHGPSFKEKTEVEPFENIEVYNLMCDLLRIQPAPNNGTHGSLNHLLKVPFYEPSHAEEVSKFSVCGFANPLPTESLDCFCPHLQNSTQLEQVNQMLNLTQEEITATVKVNLPFGRPRVLQKNVDHCLLYHREYVSGFGKAMRMPMWSSYTVPQLGDTSPLPPTVPDCLRADVRVPPSESQKCSFYLADKNITHGFLYPPASNRTSDSQYDALITSNLVPMYEEFRKMWDYFHSVLLIKHATERNGVNVVSGPIFDYNYDGHFDAPDEITKHLANTDVPIPTHYFVVLTSCKNKSHTPENCPGWLDVLPFIIPHRPTNVESCPEGKPEALWVEERFTAHIARVRDVELLTGLDFYQDKV QPVSEILQLKTYLPTFETTI FOLR149 MAQRMITQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQCRPWRKNACCSINTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS HAVCR1 50MHPQVVILSLILHLADSVAGSVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVTYRKDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIVPPKVITTPIVTTVPTVTIVRTSTIVPITTIVPMTTVPITTVPITMSIPTITTVLTTMTVSTITSVPITTSIPITTSVPVITTVSTFVPPMPLPRQNHEPVATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTTDGNDTVTESSDGLWNNNQTQLFLEHSLLTANTTKGIYAGVCISVLVLLALLGVIIAKKYFFKKEVQQLSVSFSSLQIKALQNAVEKEVQAEDNIYIENSL YATD KIT 51MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVICTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPRNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCYGDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNEYMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV MET 52MKAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPNFTAETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVLEHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGSVNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSALGAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDGFMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLDAQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFNILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRSAMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRNSSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDLTIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHTLNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGWCHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICGWDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAMNKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLLTLIGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEFAVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGKNLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLNLQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNENVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDLLKLNSELNIEWKQAISSTVLGKVIVQPDQNFTGLIAGVVSISTALLLLLGFFLWLKKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVSPTTEMVSNESVDYRATFPEDQFPNSSQNGSCRQVQYPLTDMSPILTSGDSDISSPLLQNTVHIDLSALNPELVQAVQHVVIGPSSLIVHFNEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFLTEGIIMKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIRNETHNPTVKDLIGFGLQVAKGMKYLASKKFVHRDLAARNCMLDEKFTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKFTTKSDVWSFGVLLWELMTRGAPPYPDVNTFDITVYLLQGRRLLQPEYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYVHVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS MUC16 53MLKPSGLPGSSSPIRSLMTGSRSTKATPEMDSGLTGATLSPKTSTGAIVVTEHTLPFTSPDKTLASPTSSVVGRTTQSLGVMSSALPESTSRGMTHSEQRTSPSLSPQVNGTPSRNYPATSMVSGLSSPRTRTSSTEGNFTKEASTYTLTVETTSGPVTEKYTVPTETSTTEGDSTETPWDTRYIPVKITSPMKTFADSTASKENAPVSMTPAETTVIDSHIPGRTNPSFGTLYSSFLDLSPKGTPNSRGETSLELILSTTGYPFSSPEPGSAGHSRISTSAPLSSSASVLDNKISETSIFSGQSLTSPLSPGVPEARASTMPNSAIPFSMTLSNAETSAERVRSTISSLGTPSISTKQTAETILTFHAFAETMDIPSTHIAKTLASEWLGSPGTLGGTSTSALTTTSPSTTLVSEETNTHHSTSGKETEGTLNTSMTPLETSAPGEESEMTATLVPTLGFTTLDSKIRSPSQVSSSHPTRELRTTGSTSGRQSSSTAAHGSSDILRATTSSTSKASSWTSESTAQQFSEPQHTQWVETSPSMKTERPPASTSVAAPITTSVPSVVSGFTTLKTSSTKGIWLEETSADTLIGESTAGPITHQFAVPTGISMTGGSSTRGSQGTTHLLTRATASSETSADLTLATNGVPVSVSPAVSKTAAGSSPPGGTKPSYTMVSSVIPETSSLQSSAFREGTSLGLTPLNTRHPFSSPEPDSAGHTKISTSIPLLSSASVLEDKVSATSTFSHHKATSSITTGTPEISTKTKPSSAVLSSMILSNAATSPERVRNATSPLTHPSPSGEETAGSVLTLSTSAETTDSPNIHPTGTLTSESSESPSTLSLPSVSGVKTTFSSSTPSTHLFTSGEETEETSNPSVSQPETSVSRVRTTLASTSVPTPVFPTMDTWPIRSAQFSSSHLVSELRATSSTSVINSTGSALPKISHLTGTATMSQTNRDTFNDSAAPQSTTWPETSPRFKTGLPSATTIVSTSATSLSATVMVSKFTSPATSSMEATSIREPSTTILTTETTNGPGSMAVASTNIPIGKGYITEGRLDTSHLPIGTTASSETSMDFTMAKESVSMSVSPSQSMDAAGSSTPGRTSQFVDTFSDDVYHLTSREITIPRDGTSSALTPQMTATHPPSPDPGSARSTWLGILSSSPSSPTPKVIMSSTFSTQRVITSMIMDTVETSRWNMPNLPSTTSLIPSNIPTSGAIGKSTLVPLDTPSPATSLEASEGGLPTLSTYPESTNTPSIHLGAHASSESPSTIKLTMASVVKPGSYTPLTFPSIETHIHVSTARMAYSSGSSPEMTAPGETNIGSTWDPITYITTTDPKDISSAQVSTPHSVRTLRITENHPKTESATPAAYSGSPKISSSPNLTSPATKAWTITDTTEHSTQLHYTKLAEKSSGFETQSAPGPVSVVIPTSPTIGSSTLELTSDVPGEPLVLAPSEQTTITLPMATWLSTSLTEEMASTDLDISSPSSPMSTFAIFPPMSTPSHELSKSEADTSAIRNTDSTILDQHLGIRSLGRTGDLTTVPITPLTTTWTSVIEHSTQAQDTLSATMSPTHVTQSLKDQTSIPASASPSHLTEVYPELGTQGRSSSEATTFWKPSTDTLSREIETGPTNIQSTPPMDNTTTGSSSSGVTLGIAHLPIGTSSPAETSTNMALERRSSTATVSMAGTMGLLVISAPGRSISQSLGRVSSVLSESTTEGVIDSSKGSSPRLNIQGNTALSSSLEPSYAEGSQMSTSIPLTSSPTTPDVEFIGGSTFWTKEVTIVMTSDISKSSARTESSSATLMSTALGSTENTGKEKLRTASMDLPSPIPSMEVTPWISLTLSNAPNTTDSLDLSHGVHTSSAGTLATDRSLNTGVTRASRLENGSDTSSKSLSMGNSTHTSMTYTEKSEVSSSIHPRPETSAPGAETTLTSTPGNRAISLTLPFSSIPVEEVISTGITSGPDINSAPMTHSPITPPTIVWTSTGTIEQSTQPLHAVSSEKVSVQTQSTPYVNSVAVSASPTHENSVSSGSSTSSPYSSASLESLDSTISRRNAITSWLWDLTTSLPTTTWPSTSLSEALSSGHSGVSNPSSITTEFPLFSAASTSAAKQRNPETETHGPQNTAASTLNTDASSVTGLSETPVGASISSEVPLPMAITSRSDVSGLTSESTANPSLGTASSAGTKLTRTISLPTSESLVSFRMNKDPWTVSIPLGSHPTTNTETSIPVNSAGPPGLSTVASDVIDTPSDGAESIPTVSFSPSPDTEVITISHFPEKTTHSFRTISSLTHELTSRVTPIPGDWMSSAMSTKPTGASPSITLGERRTITSAAPTTSPIVLTASFTETSTVSLDNETTVKTSDILDARKTNELPSDSSSSSDLINTSIASSTMDVTKTASISPTSISGMTASSSPSLFSSDRPQVPTSTTETNTATSPSVSSNTYSLDGGSNVGGTPSTLPPFTITHPVETSSALLAWSRPVRTFSTMVSTDTASGENPTSSNSVVTSVPAPGTWTSVGSTTDLPAMGFLKTSPAGEAHSLLASTIEPATAFTPHLSAAVVTGSSATSEASLLTTSESKAIHSSPQTPTTPTSGANWETSATPESLLVVTETSDTTLTSKILVTDTILFSTVSTPPSKFPSTGTLSGASFPTLLPDTPAIPLTATEPTSSLATSFDSTPLVTIASDSLGTVPETTLTMSETSNGDALVLKTVSNPDRSIPGITIQGVTESPLHPSSTSPSKIVAPRNITYEGSITVALSTLPAGTTGSLVFSQSSENSETTALVDSSAGLERASVMPLITGSQGMASSGGIRSGSTHSTGIKTFSSLPLIMNPGEVTAMSEITTNRLTATQSTAPKGIPVKPTSAESGLLTPVSASSSPSKAFASLTTAPPTWGIPQSTLTFEFSEVPSLDTKSASLPTPGQSLNTIPDSDASTASSSLSKSPEKNPRARMMTSTKAISASSFQSTGFTETPEGSASPSMAGHEPRVPTSGTGDPRYASESMSYPDPSKASSAMTSTSLASKLITLFSTGQAARSGSSSSPISLSTEKETSFLSPTASTSRKTSLFLGPSMARQPNILVHLQTSALTLSPTSTLNMSQEEPPELTSSQTIAEEEGTTAETQTLTFTPSETPTSLLPVSSPTEPTARRKSSPETWASSISVPAKTSLVETTDGTLVTTIKMSSQAAQGNSTWPAPAEETGSSPAGTSPGSPEMSTTLKIMSSKEPSISPEIRSTVRNSPWKTPETTVPMETTVEPVTLQSTALGSGSTSISHLPTGITSPIKSPTENMLATERVSLSPSPPEAWINLYSGTPGGTRQSLATMSSVSLESPTARSITGTGQQSSPELVSKTTGMEFSMWHGSTGGTTGDTHVSLSTSSNILEDPVTSPNSVSSLTDKSKHKTETWVSTTAIPSTVLNNKIMAAEQQTSRSVDEAYSSTSSWSDQTSGSDITLGASPDVTNTLYITSTAQTTSLVSLPSGDQGITSLINPSGGKISSASSVISPSIGLETLRANVSAVKSDIAPTAGHLSQTSSPAEVSILDVTTAPTPGISTTITTMGTNSISTTTPNPEVGMSTMDSTPATERRITSTEHPSTWSSTAASDSWTVTDMISNLKVARSPGTISTMHTTSFLASSTELDSMSTPHGRITVIGTSLVTPSSDASAVKTETSTSERTLSPSDTTASTPISTFSRVQRMSISVPDILSTSWTPSSTEAEDVPVSMVSTDHASTKTDPNTPLSTFLFDSLSTLDWDTGRSLSSATATTSAPQGATTPQELTLETMISPATSQLPFSIGHITSAVTPAAMARSSGVTFSRPDPTSKKAEQTSTQLPTITSAHPGQVPRSAATTLDVIPHTAKTPDATFQRQGQTALTTEARATSDSWNEKEKSTPSAPWITEMMNSVSEDTIKEVISSSSVLRTLNILDINLESGTTSSPSWKSSPYERIAPSESTTDKEAIHPSTNTVETTGWVISSEHASHSTIPAHSASSKLTSPVVITSTREQAIVSMSTTTWPESTRARTEPNSFLTIELRDVSPYMDTSSTTQTSIISSPGSTAITKGPRTEITSSKRISSSFLAQSMRSSDSPSEAITRLSNFPAMTESGGMILAMQTSPPGATSLSAPTLDTSATASWIGTPLATTQRFTYSEKTTLFSKGPEDTSQPSPPSVEETSSSSSLVPIHATTSPSNILLTSQGHSPSSTPPVTSVFLSETSGLGKTTDMSRISLEPGTSLPPNLSSTAGEALSTYEASRDTKAIHHSADTAVINMEATSSEYSPIPGHTKPSKATSPLVTSHIMGDITSSTSVFGSSETTEIETVSSVNQGLQERSTSQVASSATETSTVITHVSSGDATTHVTKTQATFSSGTSISSPHQFITSTNTFTDVSTNPSTSLIMTESSGVTITTQTGPTGAATQGPYLLDTSTMPYLTETPLAVTPDFMQSEKTTLISKGPKDVSWTSPPSVAETSYPSSLTPFLVTTIPPATSTLQGQHTSSPVSATSVLTSGLVKTTDMLNTSMEPVTNSPQNLNNPSNEILATLAATTDIETIHPSINKAVTNMGTASSAHVLHSTLPVSSEPSTATSPMVPASSMGDALASISIPGSETTDIEGEPTSSLTAGRKENSTLQEMNSTTESNIILSNVSVGAITEATKMEVPSFDATFIPTPAQSTKFPDIFSVASSRLSNSPPMTISTHMTTTQTGSSGATSKIPLALDTSTLETSAGTPSVVTEGFAHSKITTAMNNDVKDVSQTNPPFQDEASSPSSQAPVLVTTLPSSVAFTPQWHSTSSPVSMSSVLTSSLVKTAGKVDTSLETVTSSPQSMSNTLDDISVTSAATTDIETTHPSINTVVTNVGTTGSAFESHSTVSAYPEPSKVTSPNVITSTMEDTTISRSIPKSSKTTRTETETTSSLTPKLRETSISQEITSSTETSTVPYKELTGATTEVSRTDVISSSSTSFPGPDQSTVSLDISTETNTRLSTSPIMTESAEITITTQTGPHGATSQDTFTMDPSNTTPQAGIHSAMTHGFSQLDVTILMSRIPQDVSWTSPPSVDKTSSPSSFLSSPAMTTPSLISSTLPEDKLSSPMTSLLTSGLVKITDILRTRLEPVTSSLPNFSSTSDKILATSKDSKDTKEIFPSINTEETNVKANNSGHESHSPALADSETPKATTQMVITTTVGDPAPSTSMPVHGSSETTNIKREPTYFLTPRLRETSTSQESSFPIDTSFLLSKVPIGTITEVSSTGVNSSSKISTPDHDKSTVPPDTFTGEIPRVFTSSIKTKSAEMTITTQASPPESASHSTLPLDTSTTLSQGGTHSTVTQGFPYSEVTTLMGMGPGNVSWMTTPPVEETSSVSSLMSSPAMTSPSPVSSTSPQSIPSSPLPVTALPTSVLVTTTDVLGTTSPESVTSSPPNLSSITHERPATYKDTAHTEAAMHHSTNTAVINVGTSGSGHKSQSSVLADSETSKATPLMSTTSTLGDTSVSTSTPNISQTNQIQTEPTASLSPRLRESSTSEKTSSTTETNTAFSYVPTGAITQASRTEISSSRTSISDLDRPTIAPDISTGMITRLFTSPIMIKSAEMTVITQTTTPGATSQGILPWDTSTTLFQGGTHSTVSQGFPHSEITTLRSRTPGDVSWMTTPPVEETSSGFSLMSPSMTSPSPVSSTSPESIPSSPLPVTALLTSVLVTTINVLGTTSPEPVISSPPNLSSPTQERLITYKDTAHTEAMHASMHTNTAVANVGTSISGHESQSSVPADSHTSKATSPMGITFAMGDTSVSTSTPAFFETRIQTESTSSLIPGLRDTRTSEEINTVTETSTVLSEVPITITTEVSRTEVITSSRTTISGPDHSKMSPYISTETITRLSTFPFVTGSTEMAITNQTGPIGTISQATLTLDTSSTASWEGTHSPVTQRFPHSEETTTMSRSTKGVSWQSPPSVEETSSPSSPVPLPAITSHSSLYSAVSGSSPTSALPVISLLTSGRRKTIDMLDTHSELVISSLPSASSFSGEILTSEASTNTETIHFSENTAETNMGTTNSMHKLHSSVSIHSQPSGHTPPKVTGSMMEDAIVSTSTPGSPETKNVDRDSTSPLTPELKEDSTALVMNSTTESNTVFSSVSLDAATEVSRAEVTYYDPIFMPASAQSTKSPDISPEASSSHSNSPPLTISTHKTIATQTGPSGVTSLGQLTLDTSTIATSAGTPSARTQDFVDSETTSVMNNDLNDVLKTSPFSAEEANSLSSQAPLLVTTSPSPVTSTLQEHSTSSLVSVTSVPTPTLAKITDMDTNLEPVIRSPQNLRNTLATSEATTDTHTMHPSINTAVANVGTTSSPNEFYFTVSPDSDPYKATSAVVITSTSGDSIVSTSMPRSSAMKKIESETTFSLIFRLRETSTSQKIGSSSDTSTVFDKAFTAATTEVSRTELTSSSRTSIQGTEKPTMSPDTSTRSVTMLSTFAGLTKSEERTIATQTGPHRATSQGTLTWDTSITTSQAGTHSAMTHGFSQLDLSTLTSRVPEYISGTSPPSVEKTSSSSSLLSLPAITSPSPVPTTLPESRPSSPVHLTSLPTSGLVKITDMLASVASLPPNLGSTSHKIPTTSEDIKDTEKMYPSTNIAVTNVGTTTSEKESYSSVPAYSEPPKVISPMVISFNIRDTIVSTSMPGSSEITRIEMESTFSLAHGLKGTSTSQDPIVSTEKSAVLHKLITGATETSRTEVASSRRTSIPGPDHSTESPDISTEVIPSLPISLGITESSNMTIITRTGPPLGSTSQGTFTLDTPTTSSRAGTHSMATQEFPHSEMTTVMNKDPEILSWTIPPSIEKTSFSSSLMPSPAMTSPPVSSTLPKTIHTTPSPMTSLLTPSLVMTTDTLGTSPEPTTSSPPNLSSTSHEILTTDEDTTAIEAMHPSTSTAATNVETTSSGHGSQSSVLADSEKTKATAPMDTTSTMGHTTVSTSMSVSSETTKIKRESTYSLTPGLRETSISQNASFSTDTSIVLSEVPIGTTAEVSRTEVISSGRTSIPGPSQSTVLPEISTRTMTRLFASPTMTESAEMTIPTQTGPSGSTSQDTLILDTSTIKSQAKTHSTLIQRFPHSEMTTLMSRGPGDMSWQSSPSLENPSSLPSLLSLPATTSPPPISSTLPVTISSSPLPVTSLLTSSPVTTTDMLHTSPELVTSSPPKLSHTSDERLTTGKDTTNTEAVHPSTNTAASNVEIPSSGHESPSSALADSETSKATSPMFITSTQEDTTVAISTPHFLETSRIQKESISSLSPKLRETGSSVETSSAIETSAVLSEVSIGATTEISRTEVISSSRTSISGSAESTMLPEISTTRKIIKFPTSPILAESSEMTIKTQTSPPGSTSESTFTLDTSTTPSLVITHSTMTQRLPHSEITTLVSRGAGDVPRPSSLPVEETSPPSSQLSLSAMISPSPVSSTLPASSHSSSASVTSLLTPGQVKTTEVLDASAEPETSSPPSLSSTSVEILATSEVTTDTEKIHPFSNTAVTKVGTSSSGHESPSSVLPDSETTKATSAMGTISIMGDTSVSTLTPALSNTRKIQSEPASSLTTRLRETSTSEETSLATEANTVLSKVSTGATTEVSRTEAISFSRTSMSGPEQSTMSQDISIGTIPRISASSVLTESAKMTITTQTGPSESTLESTLNLNTATTPSWVETHSIVIQGFPHPEMTTSMGRGPGGVSWPSPPFVKETSPPSSPLSLPAVTSPHPVSTTFLAHIPPSPLPVTSLLTSGPATTTDILGTSTEPGTSSSSSLSTTSHERLITYKDTAHTEAVHPSTNTGGINVATTSSGYKSQSSVLADSSPMCITSTMGDTSVLTSTPAFLETRRIQTELASSLTPGLRESSGSEGTSSGTKMSTVLSKVPTGATTEISKEDVISIPGPAQSTISPDISTRIVSWFSTSPVMTESAEITMNTHTSPLGATTQGTSTLDTSSITSLTMTHSTISQGFSHSQMSTLMRRGPEDVSWMSPPLLEKTRPSFSLMSSPATTSPSPVSSTLPESISSSPLPVTSLLTSGLAKTTDMLHKSSEPVTNSPANLSSTSVEILATSEVTTDTEKTHPSSNRTVTDVGISSSGHESTSFVLADSQTSKVISPMVITSTMEDTSVSTSTPGFFETSRIQTEPTSSLTLGLRKTSSSEGTSLATEMSTVLSGVPTGATAEVSRTEVISSSRTSISGFAQLTVSPETSTETITRLPTSSIMTESAEMMIKTQTDPPGSTPESTHTVDISTTPNWVETHSTVTQRFSHSEMTILVSRSPGDMLWPSQSSVEETSSASSLLSLPATTSPSPVSSTLVEDFPSASLPVISLLNPGLVITTDRMGISREPGISSTSNLSSTSHERLTTLEDTVDTEDMQPSTHTAVINVRTSISGHESQSSVLSDSETPKATSPMGTTYTMGETSVSISTSDFFETSRIQIEPTSSLTSGLRETSSSERISSATEGSTVLSEVPSGATTEVSRTEVISSRGTSMSGPDQFTISPDISTEAITRLSTSPIMTESAESAITIETGSPGATSEGTLILDTSITTFWSGTHSTASPGFSHSEMTTLMSRTPGDVPWPSLPSVEEASSVSSSLSSPAMTSTSFFSTLPESISSSPHPVTALLTLGPVKITDMLRTSSEPETSSPPNLSSTSAEILATSEVTKDREKIHPSSNTPVVNVGTVIYKHLSPSSVLADLVTTKPTSPMATTSTLGNTSVSTSTPAFPETMMTQPTSSLTSGLREISTSQETSSATERSASLSGMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTETITRISTPLITTGSAEMTITPKTGHSGASSQGTFTLDTSSRASWPGTHSAATHRSPHSGMTTPMSRGPEDVSWPSRPSVEKTSPPSSLVSLSAVISPSPLYSTPSESSHSSPLRVISLFTPVMMKTTDMLDTSLEPVITSPPSMNITSDESLATSKATMETEAIQLSENTAVTQMGTISARQEFYSSYPGLPEPSKVISPVVISSTIKDIVSTTIPASSEITRIEMESTSTLTPTPRETSTSQEIHSATKPSTVPYKALTSATIEDSMTQVMSSSRGPSPDQSTMSQDISTEVITRLSTSPIKTESTEMTITTQTGSPGATSRGILTLDTSTTFMSGTHSTASQGFSHSQMTALMSRTPGDVPWLSHPSVEEASSASFSLSSPVMTSSSPVSSTLPDSIHSSSLPVTSLLTSGLVKTTELLGTSSEPETSSPPNLSSTSAEILAITEVTTDTEKLEMTNVVTSGYTHESPSSVLADSVTTKATSSMGITYPTGDTNVLTSTPAFSDTSRIQTKSKLSLTPGLMETSISEETSSATEKSTVLSSVPTGATTEVSRTEAISSSRTSIPGPAQSTMSSDTSMETITRISTPLTRKESTDMAITPKTGPSGATSQGTFTLDSSSTASWPGTHSATTQRFPQSVVTIPMSRGPEDVSWPSPLSVEKNSPPSSLVSSSSVISPSPLYSTPSGSSHSSPVPVTSLFTSIMMKATDMLDASLEPETTSAPNMNITSDESLAASKATTETEAIHVFENTAASHVETTSATEELYSSSPGFSEPTKVISPVVISSSIRDNMVSTIMPGSSGITRIEIESMSSLTPGLRETRTSQDITSSTETSTVLYKMPSGATPEVSRTEVMPSSRTSIPGPAQSTMSLDISDEVVIRLSTSPIMTESAEITITTQTGYSLATSQVTLPLGTSMTFLSGTHSTMSQGLSHSEMTNLMSRGPESLSWTSPRFVETTRSSSSLTSLPLITSLSPVSSILLDSSPSSPLPVISLILPGLVKTTEVLDTSSEPKTSSSPNLSSTSVEIPATSEIMTDTEKIHPSSNTAVAKVRTSSSVHESHSSVLADSETTITIPSMGITSAVDDTTVFTSNPAFSETRRIPTEPTFSLTPGFRETSTSEETTSITETSAVLYGVPTSATTEVSMTEIMSSNRIHIPDSDQSTMSPDIITEVITRLSSSSMMSESTQMTITTQKSSPGATAQSTLTLATTTAPLARTHSTVPPRFLHSEMTTLMSRSPENPSWKSSLFVEKTSSSSSLLSLPVITSPSVSSTLPQSIPSSSFSVTSLLTPGMVKTTDTSTEPGTSLSPNLSGTSVEILAASEVTTDTEKIHPSSSMAVTNVGTTSSGHELYSSVSIHSEPSKATYPVGTPSSMAETSISTSMPANFETTGFEAEPFSHLTSGFRKTNMSLDTSSVTPTNTPSSPGSTHLLQSSKTDFTSSAKTSSPDWPPASQYTEIPVDIITPFNASPSITESTGITSFPESRFTMSVTESTHHLSTDLLPSAETISTGTVMPSLSEAMTSFATTGVPRAISGSGSPFSRTESGPGDATLSTIAESLPSSTPVPFSSSTFITTDSSTIPALHEITSSSATPYRVDTSLGTESSTTEGRLVMVSTLDTSSQPGRTSSSPILDTRMTESVELGTVISAYQVPSLSTRLTRIDGIMEHITKIPNEAAHRGTIRPVKGPQTSTSPASPKGLHIGGTKRMETTITALKTITTALKTTSRATLITSVYTPTLGTLTPLNASMQMASTIPTEMMITTPYVFPDVPETTSSLATSLGAETSTALPRTTPSVFNRESETTASLVSRSGAERSPVIQTLDVSSSEPDTTASWVIHPAETIPTVSKTTPNFFHSELDTVSSTATSHGADVSSAIPTNISPSELDALTPLVTISGTDTSTTFPTLIKSPHETETRTTWLTHPAETSSTIPRTIPNFSHHESDATPSIATSPGAETSSAIPIMTVSPGAEDLVISQVISSGTDRNMTIPTLTLSPGEPKTIASLVTHPEAQTSSAIPTSTISPAVSRLVISMVISLAAKTSTTNRALTNSPGEPATTVSLVTHPAQTSPTVPWITSIFFHSKSDTTPSMITSHGAESSSAVPIPTVSTEVPGVVTPLVISSRAVISTTIPILTLSPGEPETTPSMATSHGEEASSAIPTPTVSPGVPGVVTSLVISSRAVISTTIPILTFSLGEPETTPSMATSHGTEAGSAVPTVLPEVPGMVISLVASSRAVISTTLPTLTLSPGEPETTPSMATSHGAEASSTVPTVSPEVPGVVTSLVTSSSGVNSTSIPTLILSPGELETTPSMATSHGAEASSAVPIPTVSPGVSGVVTPLVISSRAVISTTIPILTLSSSEPETTPSMATSHGVEASSAVLIVSPEVPGMVISLVISSRAVTSTTIPTLTISSDEPETTTSLVTHSEAKMISAIPTLAVSPTVQGLVTSLVTSSGSETSAFSNLTVASSQPETIDSWVAHPGTEASSVVPTLIVSTGEPFTNISLVTHPAESSSTLPRITSRFSHSELDTMPSTVISPEAESSSAISTTISPGIPGVLTSLVISSGRDISATFPTVPESPHESEATASWVTHPAVISTIVPRTTPNYSHSEPDTTPSIATSPGAEATSDFPTITVSPDVPDMVTSQVTSSGTDTSITIPTLTLSSGEPETITSFITYSETHISSAIPTLPVSPGASKMLTSLVISSGTDSTTIFPTLTETPYEPETTAIQLIHPAETNTMVPRTTPKFSHSKSDTTLPVAITSPGPEASSAVSTTTISPDMSDLVTSLVPSSGTDTSTTFPTLSETPYEPETTATWLTHPAETSTIVSGTIPNFSHRGSDTAPSMVISPGVDTRSGVPITTIPPSIPGVVISQVISSATDTSTAIPTLIPSPGEPETTASSATHPGTQTGFTVPIRTVPSSEPDTMASWVTHPPQTSTPVSRTTSSFSHSSPDATPVMATSPRTEASSAVLITISPGAPEMVISQITSSGAATSTIVPILTHSPGMPETTALLSTHPRTETSKTFPASTVFPQVSETTASLTIRPGAETSTALPTQTTSSLFTLLVTGTSRVDLSPTASPGVSAKTAPLSTHPGTETSTMIPTSTLSLGLLETTGLLATSSSAETSTSTLTLTVSPAVSGLSSASITTDKPQTVTSWNTETSPSVISVGPPEFSRTVTGITMTLIPSEMPTPPKTSHGEGVSPITILRTTMVEATNLATTGSSPTVAKTTTTFNTLAGSLFTPLTTPGMSTLASESVISRTSYNHRSWISTTSSYNRRYWTPATSTPVISTFSPGISTSSIPSSTAATVPFMVPFTLNFTITNLQYEEDMRHPGSRKFNATERELQGLLKPLFRNSSLEYLYSGCRLASLRPEKDSSATAVDAICTHRPDPEDLGLDRERLYWELSNLINGIQELGPYILDRNSLYVNGFTHRSSMPTTSTPGTSTVDVGTSGTPSSSPSPTTAGPLLMPFTLNFTITNLQYEEDMRRTGSRKFNTMESVLQGLLKPLFKNTSVGPLYSGCRLTLLRPEKDGAATGVDAICTHRLDPKSPGLNREQLYWELSKLINDIEELGPYILDRNSLYVNGFTHQSSVSTTSTPGTSTVDLRTSGTPSSLSSPTIMAAGPLLVPFTLNFTITNLQYGEDMGHPGSRKFNTTERVLQGLLGPIFKNTSVGPLYSGCRLTSLRSEKDGAATGVDAICIHHLDPKSPGLNRERLYWELSQLTNGIKELGPYILDRNSLYVNGFTHRTSVPTSSTPGTSTVDLGTSGTPFSLPSPATAGPLLVLFTLNFTITNLKYEEDMHRPGSRKFNITERVLQILLGPMFKNTSVGLLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPSPTTAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQSLLGPMFKNTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKELGPYILDRNSLYVNGFTHQTSAPNTSTPGTSTVDLGTSGTPSSLPSPTSAGPLLVPFTLNFTITNLQYEEDMHHPGSRKFNITERVLQGLLGPMFKNTSVGLLYSGCRLTLLRPEKNGAATGMDAICSHRLDPKSPGLNREQLYWELSQLTHGIKELGPYTLDRNSLYVNGFTHRSSVAPTSTPGTSTVDLGTSGTPSSLPSPITAVPLLVPFTLNFTITNLQYGEDMRHPGSRKFNTTERVLQGLLGPLFKNSSVGPLYSGCRLISLRSEKDGAATGVDAICTHHLNPQSPGLDREQLYWQLSQMTNGIKELGPYTLDRNSLYVNGFTHRSSGLITSTPWTSTVDLGTSGTPSPVPSPTTTGPLLVPFTLNFTITNLQYEENMGHPGSRKFNITESVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKDGVATRVDAICTHRPDPKIPGLDRQQLYWELSQLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSTPGTFTVQPETSETPSSLPGPTATGPVLLPFTLNFTITNLQYEEDMRRPGSRKFNTTERVLQGLLMPLFKNTSVSSLYSGCRLILLRPEKDGAATRVDAVCTHRPDPKSPGLDRERLYWKLSQLTHGITELGPYTLDRHSLYVNGFTHQSSMTTTRTPDTSTMHLATSRTPASLSGPMTASPLLVLFTINFTITNLRYEENMHHPGSRKFNITERVLQGLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAATKVDAICTYRPDPKSPGLDREQLYWELSQLTHSITELGPYTLDRDSLYVNGFTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPSAASPLLVLFTLNFTITNLRYEENMQHPGSRKFNTTERVLQGLLRSLFKSTSVGPLYSGCRLTLLRPEKDGTATGVDAICTHHPDPKSPRLDREQLYWELSQLTHNITELGPYALDNDSLFVNGFTHRSSVSTTSTPGTPTVYLGASKTPASIFGPSAASHLLILFTLNFTITNLRYEENMWPGSRKFNTTERVLQGLLRPLFKNTSVGPLYSGCRLTLLRPEKDGEATGVDAICTHRPDPTGPGLDREQLYLELSQLTHSITELGPYILDRDSLYVNGFTHRSSVPTTSTGVVSEEPFTLNFTINNLRYMADMGQPGSLKFNITDNVMQHLLSPLFQRSSLGARYTGCRVIALRSVKNGAETRVDLLCTYLQPLSGPGLPIKQVFHELSQQTHGITRLGPYSLDKDSLYLNGYNEPGPDEPPTTPKPATTFLPPLSEATTAMGYHLKTLTLNFTISNLQYSPDMGKGSATFNSTEGVLQHLLRPLFQKSSMGPFYLGCQLISLRPEKDGAATGVDTTCTYHPDPVGPGLDIQQLYWELSQLTHGVTQLGFYVLDRDSLFINGYAPQNLSIRGEYQINFHIVNWNLSNPDPTSSEYITLLRDIQDKVTTLYKGSQLHDTFRFCLVTNLTMDSVLVTVKALFSSNLDPSLVEQVFLDKTLNASFHWLGSTYQLVDIHVTEMESSVYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRNIEDALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFSPLARRVDRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLTGNSDLPFWAVILIGLAGLLGVITCLICGVLVTTRRRKKEGEYNVQ QQCPGYYQSHLDLEDLQ SLC39A654 MARKLSVILILTFALSVTNPLHELKAAAFPQTTEKISPNWESGINVDLAISTRQYHLQQLFYRYGENNSLSVEGFRKLLQNIGIDKIKRIHIHHDHDHHSDHEHHSDHERHSDHEHHSEHEHHSDHDHHSHHNHAASGKNKRKALCPDHDSDSSGKDPRNSQGKGAHRPEHASGRRNVKDSVSASEVISTVYNTVSEGTHFLETIETPRPGKLFPKDVSSSTPPSVTSKSRVSRLAGRKTNESVSEPRKGFMYSRNTNENPQECFNASKLLTSHGMGIQVPLNATEFNYLCPAIINQIDARSCLIHTSEKKAEIPPKTYSLQIAWVGGFIAISIISFLSLLGVILVPLMNRVFFKFLLSFLVALAVGTLSGDAFLHLLPHSHASHHHSHSHEEPAMEMKRGPLFSHLSSQNIEESAYFDSTWKGLTALGGLYFMFLVEHVLTLIKQFKDKKKKNQKKPENDDDVEIKKQLSKYESQLSTNEEKVDTDDRTEGYLRADSQEPSHFDSQQPAVLEEEEVMIAHAHPQEVYNEYVPRGCKNKCHSHFHDTLGQSDDLIHHHHDYHHILHHHHHQNHHPHSHSQRYSREELKDAGVATLAWMVIMGDGLHNFSDGLAIGAAFTEGLSSGLSTSVAVFCHELPHELGDFAVLLKAGMTVKQAVLYNALSAMLAYLGMATGIFIGHYAENVSMWIFALTAGLFMYVALVDMVPEMLHNDASDHGCSRWGYFFLQNAGMLLGFGIMLLISIFEHKIVFRINF SLC44A4 55MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFILGYIVVGIVAWLYGDPRQVLYPRNSTGAYCGMGENKDKPYLLYFNIFSCILSSNIISVAENGLQCPTPQVCVSSCPEDPWTVGKNEFSQTVGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGRCFPWINVIPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDFAQSWYWILVALGVALVLSLLFILLLRLVAGPLVLVLILGVLGVLAYGIYYCWEEYRVLRDKGASISQLGFTTNLSAYQSVQETWLAALIVLAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLVTFVLLLICIAYWAMTALYLATSGQPQYVLWASNISSPGCEKVPINTSCNPTAHLVNSSCPGLMCVFQGYSSKGLIQRSVFNLQIYGVLGLFWTLNWVLALGQCVLAGAFASFYWAFHKPQDIPTFPLISAFIRTLRYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCCFKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNIVRVVVLDKVTDLLLFFGKLLVVGGVGVLSFFFFSGRIPGLGKDFKSPHLNYYWLPIMTSILGAYVIASGFFSVFGMCVDTLFLCFLEDLERNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK STEAP1 56MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLHLHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTLLREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVIAAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSLSYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIVGLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHALIFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRK KILKIRHGWEDVTKINKTEICSQL

In various embodiments, an ADC disclosed herein may comprise any set ofheavy and light chain variable domains listed in the tables above, orthe set of six CDR sequences from the heavy and light chain set, e.g.,by transplanting the six CDRs into a chosen human donor antibodyframework. In various embodiments, an ADC disclosed herein may compriseamino acid sequences that are homologous to the sequences listed in thetables above, so long as the ADC retains the ability to bind to itstarget cancer antigen (e.g., with a K_(D) of less than 1×10⁻⁸M) andretains one or more functional properties of the ADCs disclosed herein(e.g., ability to internalize, modulate RNA splicing, inhibit cellgrowth, etc.).

In some embodiments, the ADC further comprises human heavy and lightchain constant domains or fragments thereof. For instance, the ADC maycomprise a human IgG heavy chain constant domain (such as an IgG1) and ahuman kappa or lambda light chain constant domain. In variousembodiments, the antibody or antigen binding fragment of the describedADCs comprises a human immunoglobulin G subtype 1 (IgG1) heavy chainconstant domain with a human Ig kappa light chain constant domain.

In various other embodiments, the target cancer antigen for an ADC ishuman epidermal growth factor receptor 2 (HER2).

In various embodiments, the anti-HER2 antibody or antigen bindingfragment thereof comprises three heavy chain CDRs and three light chainCDRs as follows: heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:1,heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, heavy chain CDR3(HCDR3) consisting of SEQ ID NO:3; light chain CDR1 (LCDR1) consistingof SEQ ID NO:4, light chain CDR2 (LCDR2) consisting of SEQ ID NO:5, andlight chain CDR3 (LCDR3) consisting of SEQ ID NO:6, as defined by theKabat numbering system.

In various embodiments, the anti-HER2 antibody or antigen bindingfragment thereof comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:19, and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:20. In some embodiments,the anti-HER2 antibody or antigen binding fragment thereof comprises theheavy chain variable region amino acid sequence of SEQ ID NO:19 and thelight chain variable region amino acid sequence of SEQ ID NO:20, orsequences that are at least 95% identical to the disclosed sequences. Insome embodiments, the anti-HER2 antibody or antigen binding fragmentthereof has a heavy chain variable region amino acid sequence that is atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO:19 and/or a light chain variable region amino acid sequence thatis at least 96%, at least 97%, at least 98%, or at least 99% identicalto SEQ ID NO:20.

In various embodiments, the anti-HER2 antibody or antigen bindingfragment thereof is an internalizing antibody or internalizing antigenbinding fragment. In various embodiments, the anti-HER2 antibodycomprises a human IgG1 heavy chain constant domain and a human Ig kappalight chain constant domain.

In various embodiments, the anti-HER2 antibody comprises the heavy chainamino acid sequence of SEQ ID NO:19 or a sequence that is at least 95%identical to SEQ ID NO:19, and the light chain amino acid sequence ofSEQ ID NO:20 or a sequence that is at least 95% identical to SEQ IDNO:20. In particular embodiments, the anti-HER2 antibody comprises theheavy chain amino acid sequence of SEQ ID NO:19 and the light chainamino acid sequence of SEQ ID NO:20, or sequences that are at least 95%identical to the disclosed sequences. In some embodiments, the anti-HER2antibody has a heavy chain amino acid sequence that is at least 96%, atleast 97%, at least 98%, or at least 99% identical to SEQ ID NO:19 and alight chain amino acid sequence that is at least 96%, at least 97%, atleast 98%, or at least 99% identical to SEQ ID NO:20. In variousembodiments, the anti-HER2 antibody is trastuzumab, or an antigenbinding fragment thereof.

In various embodiments, the anti-HER2 antibody or antigen bindingfragment thereof comprises the three heavy chain CDRs and three lightchain CDRs of trastuzumab or wherein the CDRs include no more than one,two, three, four, five, or six amino acid additions, deletions orsubstitutions of HCDR1 (SEQ ID NO:1), HCDR2 (SEQ ID NO:2), HCDR3 (SEQ IDNO:3); LCDR1 (SEQ ID NO:4), LCDR2 (SEQ ID NO:5), and LCDR3 (SEQ IDNO:6).

In various other embodiments, the target cancer antigen for an ADC ishuman syndecan-1 (CD138).

In various embodiments, the anti-CD138 antibody or antigen bindingfragment thereof comprises three heavy chain CDRs and three light chainCDRs as follows: heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:7,heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:8, heavy chain CDR3(HCDR3) consisting of SEQ ID NO:9; light chain CDR1 (LCDR1) consistingof SEQ ID NO:10, light chain CDR2 (LCDR2) consisting of SEQ ID NO:11,and light chain CDR3 (LCDR3) consisting of SEQ ID NO:12, as defined bythe Kabat numbering system.

In various embodiments, the anti-CD138 antibody or antigen bindingfragment thereof comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:21, and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:22. In some embodiments,the anti-CD138 antibody or antigen binding fragment thereof comprisesthe heavy chain variable region amino acid sequence of SEQ ID NO:21 andthe light chain variable region amino acid sequence of SEQ ID NO:22, orsequences that are at least 95% identical to the disclosed sequences. Insome embodiments, the anti-CD138 antibody or antigen binding fragmentthereof has a heavy chain variable region amino acid sequence that is atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO:21 and/or a light chain variable region amino acid sequence thatis at least 96%, at least 97%, at least 98%, or at least 99% identicalto SEQ ID NO:22.

In various embodiments, the anti-CD138 antibody or antigen bindingfragment thereof is an internalizing antibody or internalizing antigenbinding fragment. In various embodiments, the anti-CD138 antibodycomprises a murine IgG2a heavy chain constant domain and a murine Igkappa light chain constant domain. In various embodiments, theanti-CD138 antibody comprises a human IgG2a heavy chain constant domainand a human Ig kappa light chain constant domain.

In various embodiments, the anti-CD138 antibody comprises the heavychain amino acid sequence of SEQ ID NO:21 or a sequence that is at least95% identical to SEQ ID NO:21, and the light chain amino acid sequenceof SEQ ID NO:22 or a sequence that is at least 95% identical to SEQ IDNO:22. In particular embodiments, the anti-CD138 antibody comprises theheavy chain amino acid sequence of SEQ ID NO:21 and the light chainamino acid sequence of SEQ ID NO:22, or sequences that are at least 95%identical to the disclosed sequences. In some embodiments, theanti-CD138 antibody has a heavy chain amino acid sequence that is atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO:21 and a light chain amino acid sequence that is at least 96%, atleast 97%, at least 98%, or at least 99% identical to SEQ ID NO:22. Invarious embodiments, the anti-CD138 antibody is B-B4, or an antigenbinding fragment thereof.

In various embodiments, the anti-CD138 antibody or antigen bindingfragment thereof comprises the three heavy chain CDRs and three lightchain CDRs of B-B4 or wherein the CDRs include no more than one, two,three, four, five, or six amino acid additions, deletions orsubstitutions of HCDR1 (SEQ ID NO:7), HCDR2 (SEQ ID NO:8), HCDR3 (SEQ IDNO:9); LCDR1 (SEQ ID NO:10), LCDR2 (SEQ ID NO:11), and LCDR3 (SEQ IDNO:12).

In various other embodiments, the target cancer antigen for an ADC ishuman ephrin type-A receptor 2 (EPHA2).

In various embodiments, the anti-EPHA2 antibody or antigen bindingfragment thereof comprises three heavy chain CDRs and three light chainCDRs as follows: heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:13,heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:14, heavy chain CDR3(HCDR3) consisting of SEQ ID NO:15; light chain CDR1 (LCDR1) consistingof SEQ ID NO:16, light chain CDR2 (LCDR2) consisting of SEQ ID NO:17,and light chain CDR3 (LCDR3) consisting of SEQ ID NO:18, as defined bythe Kabat numbering system.

In various embodiments, the anti-EPHA2 antibody or antigen bindingfragment thereof comprises a heavy chain variable region comprising theamino acid sequence of SEQ ID NO:23, and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:24. In some embodiments,the anti-EPHA2 antibody or antigen binding fragment thereof comprisesthe heavy chain variable region amino acid sequence of SEQ ID NO:23 andthe light chain variable region amino acid sequence of SEQ ID NO:24, orsequences that are at least 95% identical to the disclosed sequences. Insome embodiments, the anti-EPHA2 antibody or antigen binding fragmentthereof has a heavy chain variable region amino acid sequence that is atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO:23 and/or a light chain variable region amino acid sequence thatis at least 96%, at least 97%, at least 98%, or at least 99% identicalto SEQ ID NO:24.

In various embodiments, the anti-EPHA2 antibody or antigen bindingfragment thereof is an internalizing antibody or internalizing antigenbinding fragment. In various embodiments, the anti-EPHA2 antibodycomprises a human IgG1 heavy chain constant domain and a human Ig kappalight chain constant domain.

In various embodiments, the anti-EPHA2 antibody comprises the heavychain amino acid sequence of SEQ ID NO:23 or a sequence that is at least95% identical to SEQ ID NO:23, and the light chain amino acid sequenceof SEQ ID NO:24 or a sequence that is at least 95% identical to SEQ IDNO:24. In particular embodiments, the anti-EPHA2 antibody comprises theheavy chain amino acid sequence of SEQ ID NO:23 and the light chainamino acid sequence of SEQ ID NO:24, or sequences that are at least 95%identical to the disclosed sequences. In some embodiments, theanti-EPHA2 antibody has a heavy chain amino acid sequence that is atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO:23 and a light chain amino acid sequence that is at least 96%, atleast 97%, at least 98%, or at least 99% identical to SEQ ID NO:24. Insome embodiments, the anti-EPHA2 antibody comprises a heavy chainencoded by the nucleotide sequence of SEQ ID NO:23; and a light chainencoded by the nucleotide sequence of SEQ ID NO:24. In variousembodiments, the anti-EPHA2 antibody is 101, or an antigen bindingfragment thereof.

In various embodiments, the anti-EPHA2 antibody or antigen bindingfragment thereof comprises the three heavy chain CDRs and three lightchain CDRs of 101 or wherein the CDRs include no more than one, two,three, four, five, or six amino acid additions, deletions orsubstitutions of HCDR1 (SEQ ID NO:13), HCDR2 (SEQ ID NO:14), HCDR3 (SEQID NO:15); LCDR1 (SEQ ID NO:16), LCDR2 (SEQ ID NO:17), and LCDR3 (SEQ IDNO:18).

In various embodiments, amino acid substitutions are of single residues.Insertions usually will be on the order of from about 1 to about 20amino acid residues, although considerably larger insertions may betolerated as long as biological function is retained (e.g., binding to atarget antigen). Deletions usually range from about 1 to about 20 aminoacid residues, although in some cases deletions may be much larger.Substitutions, deletions, insertions, or any combination thereof may beused to arrive at a final derivative or variant. Generally, thesechanges are done on a few amino acids to minimize the alteration of themolecule, particularly the immunogenicity and specificity of the antigenbinding protein. However, larger changes may be tolerated in certaincircumstances. Conservative substitutions are generally made inaccordance with the following chart depicted as Table 6.

TABLE 6 Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inTable 6. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general may produce thegreatest changes in the polypeptide's properties are those in which (a)a hydrophilic residue, e.g., seryl or threonyl, is substituted for (orby) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valylor alanyl; (b) a cysteine or proline is substituted for (or by) anyother residue; (c) a residue having an electropositive side chain, e.g.,lysyl, arginyl, or histidyl, is substituted for (or by) anelectronegative residue, e.g., glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g., phenylalanine, is substituted for (orby) one not having a side chain, e.g., glycine.

In various embodiments where variant antibody sequences are used in anADC, the variants typically exhibit the same qualitative biologicalactivity and will elicit the same immune response, although variants mayalso be selected to modify the characteristics of the antigen bindingproteins as needed. Alternatively, the variant may be designed such thatthe biological activity of the antigen binding protein is altered. Forexample, glycosylation sites may be altered or removed.

Various antibodies may be used with the ADCs used herein to targetcancer cells. As shown below, the linker-payloads in the ADCs disclosedherein are surprisingly effective with different tumor antigen-targetingantibodies. Suitable antigens expressed on tumor cells but not healthycells, or expressed on tumor cells at a higher level than on healthycells, are known in the art, as are antibodies directed against them.These antibodies may be used with the linkers and splicing modulatorpayloads disclosed herein. In some embodiments, the antibody or antigenbinding fragment targets HER2, and the HER2-targeting antibody orantigen binding fragment is trastuzumab. In some embodiments, theantibody or antigen binding fragment targets CD138, and theCD138-targeting antibody or antigen binding fragment is B-B4. In someembodiments, the antibody or antigen binding fragment targets EPHA2, andthe EPHA2-targeting antibody or antigen binding fragment is 101. In someembodiments, while the disclosed linkers and splicing modulator payloadsare surprisingly effective with several different tumor-targetingantibodies, HER2-targeting antibodies such as trastuzumab,CD138-targeting antibodies such as B-B4, and EPHA2-targeting antibodiessuch as 101 provided particularly improved drug:antibody ratio,aggregation level, stability (i.e., in vitro and in vivo stability),tumor targeting (i.e., cytotoxicity, potency), and/or treatmentefficacy. Improved treatment efficacy can be measured in vitro or invivo, and may include reduced tumor growth rate and/or reduced tumorvolume.

In certain embodiments, alternate antibodies to the same targets orantibodies to different antigen targets are used and provide at leastsome of the favorable functional properties described above (e.g.,improved stability, improved tumor targeting, improved treatmentefficacy, etc.). In some embodiments, some or all of these favorablefunctional properties are observed when the disclosed linkers andsplicing modulator payloads are conjugated to an alternate HER2-,CD138-, or EPHA2-targeting antibody or antigen binding fragment. In someother embodiments, some or all of these favorable functional propertiesare observed when the disclosed linkers and splicing modulator payloadsare conjugated to a HER2-targeting antibody or antigen binding fragment.In some embodiments, the antibody or antigen binding fragment targetsHER2. In some embodiments, the HER2-targeting antibody or antigenbinding fragment is trastuzumab. In some other embodiments, some or allof these favorable functional properties are observed when the disclosedlinkers and splicing modulator payloads are conjugated to aCD138-targeting antibody or antigen binding fragment. In someembodiments, the antibody or antigen binding fragment targets CD138. Insome embodiments, the CD138-targeting antibody or antigen bindingfragment is B-B4. In some other embodiments, some or all of thesefavorable functional properties are observed when the disclosed linkersand splicing modulator payloads are conjugated to an EPHA2-targetingantibody or antigen binding fragment. In some embodiments, the antibodyor antigen binding fragment targets EPHA2. In some embodiments, theEPHA2-targeting antibody or antigen binding fragment is 101.

Linkers

In various embodiments, the linker in an ADC is stable extracellularlyin a sufficient manner to be therapeutically effective. In someembodiments, the linker is stable outside a cell, such that the ADCremains intact when present in extracellular conditions (e.g., prior totransport or delivery into a cell). The term “intact,” used in thecontext of an ADC, means that the antibody or antigen binding fragmentremains attached to the drug moiety (e.g., the splicing modulator). Asused herein, “stable,” in the context of a linker or ADC comprising alinker, means that no more than 20%, no more than about 15%, no morethan about 10%, no more than about 5%, no more than about 3%, or no morethan about 1% of the linkers (or any percentage in between) in a sampleof ADC are cleaved (or in the case of an overall ADC are otherwise notintact) when the ADC is present in extracellular conditions. In someembodiments, the linkers and/or ADCs disclosed herein are surprisinglystable compared to alternate linkers and/or ADCs with alternate linkersand/or splicing modulator payloads. In some embodiments, the ADCsdisclosed herein can remain intact for more than about 48 hours, morethan 60 hours, more than about 72 hours, more than about 84 hours, ormore than about 96 hours.

Whether a linker is stable extracellularly can be determined, forexample, by including an ADC in plasma for a predetermined time period(e.g., 2, 4, 6, 8, 16, 24, 48, or 72 hours) and then quantifying theamount of free drug moiety present in the plasma. Stability may allowthe ADC time to localize to target tumor cells and prevent the prematurerelease of the drug moiety, which could lower the therapeutic index ofthe ADC by indiscriminately damaging both normal and tumor tissues. Insome embodiments, the linker is stable outside of a target cell andreleases the drug moiety from the ADC once inside of the cell, such thatthe drug can bind to its target (e.g., to the SF3b spliceosome complex).Thus, an effective linker will: (i) maintain the specific bindingproperties of the antibody or antigen binding fragment; (ii) allowdelivery, e.g., intracellular delivery, of the drug moiety via stableattachment to the antibody or antigen binding fragment; (iii) remainstable and intact until the ADC has been transported or delivered to itstarget site; and (iv) allow for the therapeutic effect, e.g., cytotoxiceffect, of the drug moiety after cleavage or alternate releasemechanism.

Linkers may impact the physico-chemical properties of an ADC. As manycytotoxic agents are hydrophobic in nature, linking them to the antibodywith an additional hydrophobic moiety may lead to aggregation. ADCaggregates are insoluble and often limit achievable drug loading ontothe antibody, which can negatively affect the potency of the ADC.Protein aggregates of biologics, in general, have also been linked toincreased immunogenicity. As shown below, linkers disclosed hereinresult in ADCs with low aggregation levels and desirable levels of drugloading.

A linker may be “cleavable” or “non-cleavable” (Ducry and Stump (2010)Bioconjugate Chem. 21:5-13). Cleavable linkers are designed to releasethe drug moiety (e.g., the splicing modulator) when subjected to certainenvironment factors, e.g., when internalized into the target cell,whereas non-cleavable linkers generally rely on the degradation of theantibody or antigen binding fragment itself.

In some embodiments, the linker is a non-cleavable linker. In someembodiments, the splicing modulator drug moiety of the ADC is releasedby degradation of the antibody or antigen binding fragment.Non-cleavable linkers tend to remain covalently associated with at leastone amino acid of the antibody and the drug upon internalization by anddegradation within the target cell. Numerous exemplary non-cleavablelinkers are described herein, and others are known in the art. Exemplarynon-cleavable linkers may comprise thioether, cyclohexyl, N-succinimidyl4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC), orN-hydroxysuccinimide (NHS), one or more polyethylene glycol (PEG)moieties, e.g., 1, 2, 3, 4, 5, or 6 PEG moieties, or one or more alkylmoieties.

In some embodiments, the linker is a cleavable linker. A cleavablelinker refers to any linker that comprises a cleavable moiety. As usedherein, the term “cleavable moiety” refers to any chemical bond that canbe cleaved. Suitable cleavable chemical bonds are well known in the artand include, but are not limited to, acid labile bonds,protease/peptidase labile bonds, photolabile bonds, disulfide bonds, andesterase labile bonds. Linkers comprising a cleavable moiety can allowfor the release of the splicing modulator drug moiety from the ADC viacleavage at a particular site in the linker.

In some embodiments, the linker is cleavable under intracellularconditions, such that cleavage of the linker sufficiently releases thesplicing modulator drug moiety from the antibody or antigen bindingfragment in the intracellular environment to activate the drug and/orrender the drug therapeutically effective. In some embodiments, thesplicing modulator drug moiety is not cleaved from the antibody orantigen binding fragment until the ADC enters a cell that expresses anantigen specific for the antibody or antigen binding fragment of theADC, and the splicing modulator drug moiety is cleaved from the antibodyor antigen binding fragment upon entering the cell. In some embodiments,the linker comprises a cleavable moiety that is positioned such that nopart of the linker or the antibody or antigen binding fragment remainsbound to the splicing modulator drug moiety upon cleavage. Exemplarycleavable linkers include acid labile linkers,protease/peptidase-sensitive linkers, photolabile linkers, dimethyl-,disulfide-, or sulfonamide-containing linkers.

In some embodiments, the linker is a pH-sensitive linker, and issensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker is cleavable under acidic conditions. This cleavagestrategy generally takes advantage of the lower pH in the endosomal (pH˜5-6) and lysosomal (pH ˜4.8) intracellular compartments, as compared tothe cytosol (pH ˜7.4), to trigger hydrolysis of an acid labile group inthe linker, such as a hydrazone (Jain et al. (2015) Pharm Res32:3526-40). In some embodiments, the linker is an acid labile and/orhydrolyzable linker. For example, an acid labile linker that ishydrolyzable in the lysosome, and contains an acid labile group (e.g., ahydrazone, a semicarbazone, a thiosemicarbazone, a cis-aconitic amide,an orthoester, an acetal, a ketal, or the like) can be used. See, e.g.,U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker(1999) Pharm Therapeutics 83:67-123; Neville et al. (1989) Biol Chem.264:14653-61. Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond) (see, e.g.,U.S. Pat. No. 5,622,929).

In some embodiments, the linker is cleavable under reducing conditions.In some embodiments, the linker is cleavable in the presence of areducing agent, such as glutathione or dithiothreitol. In someembodiments, the linker is a cleavable disulfide linker or a cleavablesulfonamide linker.

In some embodiments, the linker is a cleavable disulfide linker. Avariety of disulfide linkers are known in the art, including, forexample, those that can be formed using SATA(N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT. See, e.g., Thorpe et al. (1987) Cancer Res. 47:5924-31;Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates inRadioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press,1987). See also U.S. Pat. No. 4,880,935. Disulfide linkers are typicallyused to exploit the abundance of intracellular thiols, which canfacilitate the cleavage of their disulfide bonds. The intracellularconcentrations of the most abundance intracellular thiol, reducedglutathione, are generally in the range of 1-10 nM, which is about1,000-fold higher than that of the most abundant low-molecular thiol inthe blood (i.e., cysteine) at about 5 μM (Goldmacher et al., In CancerDrug Discovery and Development: Antibody-Drug Conjugates andImmunotoxins (G. L. Phillips ed., Springer, 2013)). The intracellularenzymes of the protein disulfide isomerase family may also contribute tothe intracellular cleavage of a disulfide linker. As used herein, acleavable disulfide linker refers to any linker that comprises acleavable disulfide moiety. The term “cleavable disulfide moiety” refersto a disulfide bond that can be cleaved and/or reduced, e.g., by a thiolor enzyme.

In some embodiments, the linker is a cleavable sulfonamide linker. Asused herein, a cleavable sulfonamide linker refers to any linker thatcomprises a cleavable sulfonamide moiety. The term “cleavablesulfonamide moiety” refers to a sulfonamide group, i.e., sulfonyl groupconnected to an amine group, wherein the sulfur-nitrogen bond can becleaved.

In some embodiments, the linker may be a dendritic type linker forcovalent attachment of more than one drug moiety to an antibody orantigen binding fragment through a branching, multifunctional linkermoiety. See, e.g., Sun et al. (2002) Bioorg Med Chem Lett. 12:2213-5;Sun et al. (2003) Bioorg Med Chem. 11:1761-8. Dendritic linkers canincrease the molar ratio of drug to antibody, i.e., drug loading, whichis related to the potency of the ADC. Thus, where an antibody or antigenbinding fragment bears only one reactive cysteine thiol group, forexample, a multitude of splicing modulator drug moieties may be attachedthrough a dendritic linker. In some embodiments, the linker moiety orlinker-drug moiety may be attached to the antibody or antigen bindingfragment via reduced disulfide bridging chemistry or limited lysineutilization technology. See, e.g., Intl. Publ. Nos. WO 2013/173391 andWO 2013/173393.

In some embodiments, the linker is cleavable by a cleaving agent, e.g.,an enzyme, that is present in the intracellular environment (e.g.,within a lysosome or endosome or caveola). The linker can be, e.g., apeptide linker that is cleaved by an intracellular peptidase or proteaseenzyme, including, but not limited to, a lysosomal or endosomalprotease.

In some embodiments, the linker is a cleavable peptide linker. As usedherein, a cleavable peptide linker refers to any linker that comprises acleavable peptide moiety. The term “cleavable peptide moiety” refers toany chemical bond linking amino acids (natural or synthetic amino acidderivatives) that can be cleaved by an agent that is present in theintracellular environment. For instance, a linker may comprise avaline-alanine (Val-Ala) sequence, or a valine-citrulline (Val-Cit)sequence that is cleavable by a peptidase such as cathepsin, e.g.,cathepsin B. In some embodiments, a linker may comprise a glutamicacid-valine-citrulline (Glu-Val-Cit) sequence. In some embodiments, thelinker is an enzyme-cleavable linker and a cleavable peptide moiety inthe linker is cleavable by the enzyme. In some embodiments, thecleavable peptide moiety is cleavable by a lysosomal enzyme, e.g.,cathepsin. In some embodiments, the linker is a cathepsin-cleavablelinker. In some embodiments, the cleavable peptide moiety in the linkeris cleavable by a lysosomal cysteine cathepsin, such as cathepsin B, C,F, H, K, L, O, S, V, X, or W. In some embodiments, the cleavable peptidemoiety is cleavable by cathepsin B. An exemplary dipeptide that may becleaved by cathepsin B is valine-citrulline (Val-Cit) (Dubowchik et al.(2002) Bioconjugate Chem. 13:855-69).

In some embodiments, the linker or the cleavable peptide moiety in thelinker comprises an amino acid unit. In some embodiments, the amino acidunit allows for cleavage of the linker by a protease, therebyfacilitating release of the splicing modulator drug moiety from the ADCupon exposure to one or more intracellular proteases, such as one ormore lysosomal enzymes (Doronina et al. (2003) Nat Biotechnol.21:778-84; Dubowchik and Walker (1999) Pharm Therapeutics 83:67-123).Exemplary amino acid units include, but are not limited to, dipeptides,tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptidesinclude, but are not limited to, valine-alanine (Val-Ala),valine-citrulline (Val-Cit), alanine-asparagine (Ala-Asn),alanine-phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys),alanine-lysine (Ala-Lys), alanine-valine (Ala-Val), valine-lysine(Val-Lys), lysine-lysine (Lys-Lys), phenylalanine-citrulline (Phe-Cit),leucine-citrulline (Leu-Cit), isoleucine-citrulline (Ile-Cit),tryptophan-citrulline (Trp-Cit), and phenylalanine-alanine (Phe-Ala).Exemplary tripeptides include, but are not limited to,alanine-alanine-asparagine (Ala-Ala-Asn), glycine-valine-citrulline(Gly-Val-Cit), glycine-glycine-glycine (Gly-Gly-Gly),phenylalanine-phenylalanine-lysine (Phe-Phe-Lys), glutamicacid-valine-citrulline (Glu-Val-Cit) (see, e.g., Anami et al. (2018) NatComm. 9:2512, which is incorporated herein by reference for exemplarylinkers comprising Glu-Val-Cit), and glycine-phenylalanine-lysine(Gly-Phe-Lys). Other exemplary amino acid units include, but are notlimited to, Gly-Phe-Gly-Gly (SEQ ID NO:34), Gly-Phe-Leu-Gly (SEQ IDNO:35), Ala-Leu-Ala-Leu (SEQ ID NO:36), Phe-N⁹-tosyl-Arg, andPhe-N⁹-Nitro-Arg, as described in, e.g., U.S. Pat. No. 6,214,345. Insome embodiments, the amino acid unit in the linker comprises Val-Ala.In some embodiments, the amino acid unit in the linker comprisesVal-Cit. In some embodiments, the amino acid unit in the linkercomprises Glu-Val-Cit. An amino acid unit may comprise amino acidresidues that occur naturally and/or minor amino acids and/ornon-naturally occurring amino acid analogs, such as citrulline. Aminoacid units can be designed and optimized for enzymatic cleavage by aparticular enzyme, for example, a tumor-associated protease, a lysosomalprotease such as cathepsin B, C, D, or S, or a plasmin protease.

In some embodiments, the linker is a cleavable β-glucuronide linker. Asused herein, a cleavable β-glucuronide linker refers to any linker thatcomprises a cleavable β-glucuronide moiety. An exemplary cleavableβ-glucuronide linker comprises the structure:

The term “cleavable β-glucuronide moiety” refers to a glycosidic bondthat can be cleaved by an agent having β-glucuronidase activity. In someembodiments, the linker comprises a glycosidic bond that can be cleavedby a β-glucuronidase. A β-glucuronidase is a UDP-glucuronosyltransferase that catalyzes the hydrolysis of the glycosidic bond ofglucuronides with β-configuration.

In some embodiments, an ADC disclosed herein comprises a cleavableβ-glucuronide moiety in the linker that is cleavable by the enzyme. Insome embodiments, the cleavable β-glucuronide moiety in the linker iscleavable by a lysosomal enzyme, e.g., a β-glucuronidase. In someembodiments, the linker is a β-glucuronidase-cleavable linker. In someembodiments, the cleavable β-glucuronide moiety in the linker allows forcleavage of the linker by a β-glucuronidase after internalization of theADC, thereby facilitating release of the drug moiety from the ADC in thecellular environment.

In some embodiments, the linker in any of the ADCs disclosed herein maycomprise at least one spacer unit joining the antibody or antigenbinding fragment to the drug moiety (e.g., the splicing modulator drugmoiety). In some embodiments, a spacer unit between the antibody orantigen binding fragment and cleavable moiety, when present, joins acleavage site (e.g., a cleavable peptide moiety) in the linker to theantibody or antigen binding fragment. In some embodiments, a spacer unitbetween the drug moiety and cleavable moiety, when present, joins acleavage site (e.g., a cleavable peptide moiety) in the linker to thedrug moiety. In some embodiments, no cleavage site is present, and thespacer unit is used to link the antibody or antigen binding fragment tothe drug moiety.

In some embodiments, the linker, and/or spacer unit in the linker, issubstantially hydrophilic. A hydrophilic linker may be used to reducethe extent to which the drug may be pumped out of resistant cancer cellsthrough multiple drug resistance (MDR) or functionally similartransporters. In some embodiments, a hydrophilic linker may include oneor more polyethylene glycol (PEG) moieties, e.g., 1, 2, 3, 4, 5, or 6PEG moieties. In some embodiments, the linker comprises 2 PEG moieties.

In some embodiments, the spacer unit in the linker comprises one or morePEG moieties. In some embodiments, the spacer unit comprises one or more-(PEG)_(m)-, and m is an integer from 1 to 10 (i.e., m may be 1, 2, 3,4, 5, 6, 7, 8, 9, or 10). In some embodiments, m ranges from 1 to 10;from 2 to 8; from 2 to 6; from 2 to 5; from 2 to 4; or from 2 to 3. Insome embodiments, m is 2. In some embodiments, the spacer unit comprises(PEG)₂, (PEG)₃, (PEG)₄, (PEG)₅, (PEG)₆, (PEG)₇, (PEG)₈, (PEG)₉, or(PEG)₁₀. In some embodiments, the spacer unit comprises (PEG)₂.

In some embodiments, the spacer unit in the linker comprises an alkylmoiety. In some embodiments, the spacer unit comprises one or more—(CH₂)_(n)—, and n is an integer from 1 to 10 (i.e., n may be 1, 2, 3,4, 5, 6, 7, 8, 9, or 10). In some embodiments, n ranges from 1 to 10;from 2 to 8; from 2 to 6; from 2 to 5; from 2 to 4; or from 2 to 3. Insome embodiments, n is 2. In some embodiments, n is 5. In someembodiments, n is 6. In some embodiments, the spacer unit comprises(CH₂)₂, (CH₂)₃, (CH₂)₄, (CH₂)₅, (CH₂)₆, (CH₂)₇, (CH₂)₈, (CH₂)₉, or(CH₂)₁₀. In some embodiments, the spacer unit comprises (CH₂)₂ (“Et”).In some embodiments, the spacer unit comprises (CH₂)₆ (“Hex”). In someembodiments, the spacer unit comprises (CH₂)₂—O—(CH₂)₂ (“Et-O-Et”).

A spacer unit may be used, for example, to link the antibody or antigenbinding fragment to the drug moiety, either directly or indirectly. Insome embodiments, the spacer unit links the antibody or antigen bindingfragment to the splicing modulator drug moiety directly. In someembodiments, the antibody or antigen binding fragment and the splicingmodulator drug moiety are attached via a spacer unit comprising one ormore PEG moieties (e.g., (PEG)₂), or one or more alkyl moieties (e.g.,(CH₂)₂, (CH₂)₆, or (CH₂)₂—O—(CH₂)₂). In some embodiments, the spacerunit links the antibody or antigen binding fragment to the splicingmodulator drug moiety indirectly. In some embodiments, the spacer unitlinks the antibody or antigen binding fragment to the splicing modulatordrug moiety indirectly through a cleavable moiety (e.g., a cleavablepeptide or a cleavable β-glucuronide) and/or an attachment moiety tojoin the spacer unit to the antibody or antigen binding fragment, e.g.,a maleimide moiety.

The spacer unit, in various embodiments, attaches to the antibody orantigen binding fragment (i.e., the antibody or antigen bindingfragment) via a maleimide (Mal) moiety.

A spacer unit that attaches to the antibody or antigen binding fragmentvia a Mal is referred to herein as a “Mal-spacer unit.” The term “Mal”or “maleimide moiety,” as used herein, means a compound that contains amaleimide group and that is reactive with a sulfhydryl group, e.g., asulfhydryl group of a cysteine residue on the antibody or antigenbinding fragment. Other functional groups that are reactive withsulfhydryl groups (thiols) include, but are not limited to,iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate. In some embodiments, theMal-spacer unit is reactive with a cysteine residue on the antibody orantigen binding fragment. In some embodiments, the Mal-spacer unit isjoined to the antibody or antigen binding fragment via the cysteineresidue. In some embodiments, the Mal-spacer unit comprises a PEGmoiety. In some embodiments, the Mal-spacer unit comprises an alkylmoiety.

In certain embodiments, the linker comprises the Mal-spacer unit and acleavable peptide moiety. In some embodiments, the cleavable peptidemoiety comprises an amino acid unit. In some embodiments, the amino acidunit comprises Val-Cit. In some embodiments, the amino acid unitcomprises Val-Ala. In some embodiments, the amino acid unit comprisesGlu-Val-Cit. In some embodiments, the linker comprises the Mal-spacerunit and Val-Cit. In some embodiments, the linker comprises theMal-spacer unit and Val-Ala. In some embodiments, the linker comprisesthe Mal-spacer unit and Val-Cit, wherein the Mal-spacer unit comprises amaleimidocaproyl (MC). In some embodiments, the linker comprises theMal-spacer unit and Val-Ala, wherein the Mal-spacer unit comprises amaleimidocaproyl (MC). In some embodiments, the linker comprises theMal-spacer unit and a cleavable β-glucuronide moiety.

In some embodiments, the linker comprises the structure: Mal-spacerunit. In some embodiments, the Mal-spacer unit comprises amaleimidocaproyl (MC). In some embodiments, the linker comprises thestructure: MC. In some embodiments, the linker comprises the structure:Mal-(CH₂)₂ (“Mal-Et”). In some embodiments, the linker comprises thestructure: Mal-(CH₂)₆ (“Mal-Hex”). In some embodiments, the linkercomprises the structure: Mal-(CH₂)₂—O—(CH₂)₂ (“Mal-Et-O-Et”). In someembodiments, the linker comprises the structure: Mal-(PEG)₂. In someembodiments, the linker comprises the structure: Mal-(PEG)₂-CO.

In various embodiments, the Mal-spacer unit attaches the antibody orantigen binding fragment to a cleavable peptide moiety. In someembodiments, the linker comprises Mal-spacer unit-peptide. In someembodiments, the linker comprises the structure: Mal-spacerunit-Val-Cit. In some embodiments, the Mal-spacer unit comprises amaleimidocaproyl (MC). In some embodiments, the linker comprises thestructure: MC-Val-Cit.

In some embodiments, the linker comprises the structure: Mal-spacerunit-Val-Ala. In some embodiments, the Mal-spacer unit comprises amaleimidocaproyl (MC). In some embodiments, the linker comprises thestructure: MC-Val-Ala.

In various embodiments, the Mal-spacer unit attaches the antibody orantigen binding fragment to a cleavable β-glucuronide moiety. In someembodiments, the linker comprises Mal-spacer unit-β-glucuronide. In someembodiments, the linker comprises MC-β-glucuronide.

In various embodiments, the cleavable moiety in the linker is joineddirectly to the splicing modulator drug moiety. In other embodiments, aspacer unit is used to attach the cleavable moiety in the linker to thesplicing modulator drug moiety. In various embodiments, the splicingmodulator is attached to the cleavable moiety in the linker by a spacerunit.

A spacer unit may be “self-immolative” or “non-self-immolative.” A“non-self-immolative” spacer unit is one in which part or all of thespacer unit remains bound to the splicing modulator drug moiety uponcleavage of the linker. Examples of non-self-immolative spacer unitsinclude, but are not limited to, a glycine spacer unit and aglycine-glycine spacer unit. Non-self-immolative spacer units mayeventually degrade over time but do not readily release a linked nativedrug moiety entirely under cellular conditions. A “self-immolative”spacer unit allows for release of the native drug moiety underintracellular conditions. A “native drug” or “native drug moiety” is onewhere no part of the spacer unit or other chemical modification remainsafter cleavage/degradation of the spacer unit.

Self-immolation chemistry is known in the art and could be readilyselected for the disclosed ADCs. In various embodiments, the spacer unitattaching the cleavable moiety in the linker to the splicing modulatordrug moiety is self-immolative, and undergoes self-immolationconcurrently with or shortly before/after cleavage of the cleavablemoiety under intracellular conditions. In some embodiments, the splicingmodulator is attached to the cleavable moiety in the linker by aself-immolative spacer unit. In certain embodiments, the splicingmodulator is attached to the cleavable moiety in the linker by aself-immolative spacer unit, the cleavable moiety comprises Val-Cit, anda maleimidocaproyl (MC) joins the cleavable moiety to the antibody orantigen binding fragment. In certain embodiments, the splicing modulatoris attached to the cleavable moiety in the linker by a self-immolativespacer unit, the cleavable moiety comprises Val-Ala, and amaleimidocaproyl (MC) joins the cleavable moiety to the antibody orantigen binding fragment. In certain embodiments, the splicing modulatoris attached to the cleavable moiety in the linker by a self-immolativespacer unit, the cleavable moiety comprises Glu-Val-Cit, and amaleimidocaproyl (MC) joins the cleavable moiety to the antibody orantigen binding fragment. In certain embodiments, the splicing modulatoris joined to the antibody or antigen binding fragment via a Mal-spacerunit (e.g., MC) in the linker joined to a Val-Cit cleavable moiety and apABC or pAB self-immolative spacer unit. In certain other embodiments,the splicing modulator is joined to the antibody or antigen bindingfragment via a Mal-spacer unit (e.g., MC) in the linker joined to aVal-Ala cleavable moiety and a pABC or pAB self-immolative spacer unit.In certain other embodiments, the splicing modulator is joined to theantibody or antigen binding fragment via a Mal-spacer unit (e.g., MC) inthe linker joined to a Glu-Val-Cit cleavable moiety and a pABC or pABself-immolative spacer unit.

In certain embodiments, the self-immolative spacer unit in the linkercomprises a p-aminobenzyl unit. In some embodiments, a p-aminobenzylalcohol (pABOH) is attached to an amino acid unit or other cleavablemoiety in the linker via an amide bond, and a carbamate,methylcarbamate, or carbonate is made between the pABOH and the drugmoiety (Hamann et al. (2005) Expert Opin Ther Patents 15:1087-103). Insome embodiments, the self-immolative spacer unit is or comprisesp-aminobenzyloxycarbonyl (pABC). Without being bound by theory, it isthought that the self-immolation of pABC involves a spontaneous1,6-elimination reaction (Jain et al. (2015) Pharm Res. 32:3526-40).

In various embodiments, the structure of the p-aminobenzyloxycarbonyl(pABC) used in the disclosed ADCs is shown below:

In various embodiments, the self-immolative spacer unit attaches thecleavable moiety in the linker to the splicing modulator. In someembodiments, the self-immolative spacer unit is pABC. In someembodiments, the pABC attaches the cleavable moiety in the linker to thesplicing modulator. In some embodiments, the pABC undergoesself-immolation upon cleavage of the cleavable moiety, and the splicingmodulator is released from the ADC in its native, active form.

In some embodiments, an anti-HER2 antibody or antigen binding fragmentis joined to the splicing modulator by a linker comprisingMC-Val-Cit-pABC. In other embodiments, an anti-HER2 antibody or antigenbinding fragment is joined to the splicing modulator by a linkercomprising MC-Val-Ala-pABC.

In some embodiments, an anti-CD138 antibody or antigen binding fragmentis joined to the splicing modulator by a linker comprisingMC-Val-Cit-pABC. In other embodiments, an anti-CD138 antibody or antigenbinding fragment is joined to the splicing modulator by a linkercomprising MC-Val-Ala-pABC.

In some embodiments, an anti-EPHA2 antibody or antigen binding fragmentis joined to the splicing modulator by a linker comprisingMC-Val-Cit-pABC. In other embodiments, an anti-EPHA2 antibody or antigenbinding fragment is joined to the splicing modulator by a linkercomprising MC-Val-Ala-pABC.

In some embodiments, the pABC undergoes self-immolation upon cleavage ofa cleavable peptide moiety in the linker. In some embodiments, thecleavable peptide moiety comprises an amino acid unit. In someembodiments, the linker comprises amino acid unit-pABC. In someembodiments, the amino acid unit is Val-Cit. In some embodiments, thelinker comprises Val-Cit-pABC. In some embodiments, the amino acid unitis Val-Ala. In some embodiments, the linker comprises Val-Ala-pABC. Insome embodiments, the amino acid unit is Glu-Val-Cit. In someembodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments,the amino acid unit is Ala-Ala-Asn. In some embodiments, the linkercomprises Ala-Ala-Asn-pABC.

In some embodiments, the pABC undergoes self-immolation upon cleavage ofa cleavable β-glucuronide moiety in the linker. In some embodiments, thelinker comprises β-glucuronide-pABC.

In certain embodiments, the self-immolative spacer unit in the linkercomprises a p-aminobenzyl unit. In some embodiments, the self-immolativespacer unit in the linker comprises a p-aminobenzyl (pAB). In someembodiments, the self-immolation of pAB involves a spontaneous1,6-elimination reaction.

In various embodiments, the structure of the p-aminobenzyl (pAB) used inthe disclosed ADCs is shown below:

In various embodiments, the self-immolative spacer unit attaches thecleavable moiety in the linker to the splicing modulator. In someembodiments, the self-immolative spacer unit is pAB. In someembodiments, the pAB attaches the cleavable moiety in the linker to thesplicing modulator. In some embodiments, the pAB undergoesself-immolation upon cleavage of the cleavable moiety, and the splicingmodulator is released from the ADC in its native, active form.

In some embodiments, an anti-HER2 antibody or antigen binding fragmentis joined to the splicing modulator by a linker comprisingMC-Val-Cit-pAB. In other embodiments, an anti-HER2 antibody or antigenbinding fragment is joined to the splicing modulator by a linkercomprising MC-Val-Ala-pAB.

In some embodiments, an anti-CD138 antibody or antigen binding fragmentis joined to the splicing modulator by a linker comprisingMC-Val-Cit-pAB. In other embodiments, an anti-CD138 antibody or antigenbinding fragment is joined to the splicing modulator by a linkercomprising MC-Val-Ala-pAB.

In some embodiments, an anti-EPHA2 antibody or antigen binding fragmentis joined to the splicing modulator by a linker comprisingMC-Val-Cit-pAB. In other embodiments, an anti-EPHA2 antibody or antigenbinding fragment is joined to the splicing modulator by a linkercomprising MC-Val-Ala-pAB.

In some embodiments, the pAB undergoes self-immolation upon cleavage ofa cleavable peptide moiety in the linker. In some embodiments, thecleavable peptide moiety comprises an amino acid unit. In someembodiments, the linker comprises amino acid unit-pAB. In someembodiments, the amino acid unit is Val-Cit. In some embodiments, thelinker comprises Val-Cit-pAB. In some embodiments, the amino acid unitis Val-Ala. In some embodiments, the linker comprises Val-Ala-pAB. Insome embodiments, the amino acid unit is Glu-Val-Cit. In someembodiments, the linker comprises Glu-Val-Cit-pAB. In some embodiments,the amino acid unit is Ala-Ala-Asn. In some embodiments, the linkercomprises Ala-Ala-Asn-pAB.

In some embodiments, the pAB undergoes self-immolation upon cleavage ofa cleavable β-glucuronide moiety in the linker. In some embodiments, thelinker comprises β-glucuronide-pAB.

In some other embodiments, the splicing modulator is attached to thecleavable moiety in the linker by a non-self-immolative spacer unit. Incertain embodiments, the splicing modulator is attached to the cleavablemoiety in the linker by a non-self-immolative spacer unit, the cleavablemoiety comprises Val-Cit, and a maleimidocaproyl (MC) joins thecleavable moiety to the antibody or antigen binding fragment. In certainembodiments, the splicing modulator is attached to the cleavable moietyin the linker by a non-self-immolative spacer unit, the cleavable moietycomprises Val-Ala, and a maleimidocaproyl (MC) joins the cleavablemoiety to the antibody or antigen binding fragment.

In various aspects, the antibody or antigen binding fragment of the ADCis conjugated to the splicing modulator drug moiety via a linker,wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavableamino acid unit, and a pABC. In some embodiments, the spacer unitcomprises an alkyl moiety. In some embodiments, the Mal-spacer unitcomprises a maleimidocaproyl (MC). In some embodiments, the linkercomprises Mal-spacer unit-amino acid unit-pABC. In some embodiments, thelinker comprises MC-amino acid unit-pABC. In some embodiments, thelinker comprises MC-Val-Cit-pABC. In some embodiments, the linkercomprises MC-Val-Ala-pABC. In some embodiments, the linker comprisesMC-Glu-Val-Cit-pABC. In some embodiments, the linker comprisesMC-Ala-Ala-Asn-pABC.

In various other aspects, the antibody or antigen binding fragment ofthe ADC is conjugated to the splicing modulator drug moiety via alinker, wherein the linker comprises a Mal-spacer unit (e.g., MC), acleavable amino acid unit, and a pAB. In some embodiments, the spacerunit comprises an alkyl moiety. In some embodiments, the Mal-spacer unitcomprises a maleimidocaproyl (MC). In some embodiments, the linkercomprises Mal-spacer unit-amino acid unit-pAB. In some embodiments, thelinker comprises MC-amino acid unit-pAB. In some embodiments, the linkercomprises MC-Val-Cit-pAB. In some embodiments, the linker comprisesMC-Val-Ala-pAB. In some embodiments, the linker comprisesMC-Glu-Val-Cit-pAB. In some embodiments, the linker comprisesMC-Ala-Ala-Asn-pAB.

In various other aspects, the antibody or antigen binding fragment ofthe ADC is conjugated to the splicing modulator drug moiety via alinker, wherein the linker comprises a Mal-spacer unit (e.g., MC), acleavable β-glucuronide, and a pABC. In some embodiments, the linkercomprises Mal-spacer unit-β-glucuronide-pABC. In some embodiments, thelinker comprises MC-β-glucuronide-pABC.

In still other aspects, the antibody or antigen binding fragment of theADC is conjugated to the splicing modulator drug moiety via a linker,wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavableβ-glucuronide, and a pAB. In some embodiments, the linker comprisesMal-spacer unit-β-glucuronide-pAB. In some embodiments, the linkercomprises MC-β-glucuronide-pAB.

In various embodiments, the ADC compound has Formula (I):

Ab-(L-D)_(p)  (I)

wherein Ab is an antibody or antigen binding fragment which targets aneoplastic cell;D is a splicing modulator;L is a linker that covalently attaches Ab to D; andp is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment (Ab) ofthe ADC is conjugated to the splicing modulator drug moiety via alinker, wherein the linker is any of the linkers disclosed orincorporated by reference herein, or comprises one or more components ofany of the linkers disclosed or incorporated by reference herein.

In some embodiments, the linker comprises a cleavable moiety that ispositioned such that no part of the linker or the antibody or antigenbinding fragment remains bound to the splicing modulator after cleavage.In some embodiments, the cleavable moiety is a cleavable peptide moiety,e.g., an amino acid unit such as Val-Cit or Val-Ala. In someembodiments, the amino acid unit or linker comprises Val-Cit. In someembodiments, the amino acid unit or linker comprises Val-Ala. In someembodiments, the amino acid unit or linker comprises Glu-Val-Cit.

In some embodiments, the linker comprises at least one spacer unitjoining the antibody or antigen binding fragment to the cleavablemoiety. In some embodiments, the linker comprises at least one spacerunit joining the antibody or antigen binding fragment to the drugmoiety. In some embodiments, the spacer unit or linker comprises atleast one alkyl moiety.

In some embodiments, a spacer unit in the linker attaches to theantibody or antigen binding fragment via a Mal moiety (“Mal-spacerunit”). In some embodiments, the Mal-spacer unit comprises at least onealkyl moiety. In some embodiments, the linker comprises amaleimidocaproyl (MC). In some embodiments, the linker comprisesMal-(CH₂)₂ (“Mal-Et”). In some embodiments, the linker comprisesMal-(CH₂)₆ (“Mal-Hex”). In some embodiments, the linker comprisesMal-(CH₂)₂—O—(CH₂)₂ (“Mal-Et-O-Et”). In some embodiments, the linkercomprises Mal-(PEG)₂-CO. In some embodiments, the Mal-spacer unitattaches the antibody or antigen binding fragment to the drug moiety.

In some embodiments, the Mal-spacer unit or linker comprises Mal-(PEG)₂,Mal-(PEG)₃, Mal-(PEG)₄, Mal-(PEG)₅, Mal-(PEG)₆, Mal-(PEG)₇, orMal-(PEG)₈. In some embodiments, the Mal-spacer unit or linker comprisesMal-(PEG)₂. In some embodiments, the Mal-spacer unit or linker comprisesMal-(PEG)₂-CO, Mal-(PEG)₃-CO, Mal-(PEG)₄-CO, Mal-(PEG)₅-CO,Mal-(PEG)₆-CO, Mal-(PEG)₇-CO, or Mal-(PEG)₈-CO. In some embodiments, theMal-spacer unit or linker comprises Mal-(PEG)₂-CO. In some embodiments,the Mal-spacer unit or linker comprises Mal-(PEG)₂-CO and at least oneadditional spacer unit. In some embodiments, the Mal-(PEG)₂-CO attachesthe antibody or antigen binding fragment to the drug moiety. In someembodiments, linker comprises or consists of Mal-(PEG)₂-CO. An exampleof a “Mal-(PEG)₂-CO” linker is also referred to herein as “ADL2” or an“ADL2” linker.

In some embodiments, the Mal-spacer unit or linker comprises MC. In someembodiments, the Mal-spacer unit or linker comprises MC and at least oneadditional spacer unit. In some embodiments, the MC attaches theantibody or antigen binding fragment to the drug moiety. In someembodiments, the linker comprises or consists of MC. An example of an“MC” linker is also referred to herein as “ADL10” or an “ADL10” linker.

In some embodiments, the Mal-spacer unit or linker comprises Mal-(CH₂)₆(“Mal-Hex”). In some embodiments, the Mal-spacer unit or linkercomprises Mal-Hex and at least one additional spacer unit. In someembodiments, the Mal-Hex attaches the antibody or antigen bindingfragment to the drug moiety. In some embodiments, the linker comprisesMal-Hex. An example of a “Mal-Hex” linker is also referred to herein as“ADL12” or an “ADL12” linker.

In some embodiments, the Mal-spacer unit or linker comprises Mal-(CH₂)₂(“Mal-Et”). In some embodiments, the Mal-spacer unit or linker comprisesMal-Et and at least one additional spacer unit. In some embodiments, theMal-Et attaches the antibody or antigen binding fragment to the drugmoiety. In some embodiments, the linker comprises Mal-Et. An example ofa “Mal-Et” linker is also referred to herein as “ADL14” or an “ADL14”linker.

In some embodiments, the Mal-spacer unit or linker comprisesMal-(CH₂)₂—O—(CH₂)₂ (“Mal-Et-O-Et”). In some embodiments, the Mal-spacerunit or linker comprises Mal-Et-O-Et and at least one additional spacerunit. In some embodiments, the Mal-Et-0-Et attaches the antibody orantigen binding fragment to the drug moiety. In some embodiments, thelinker comprises Mal-Et-O-Et. An example of a “Mal-Et-O-Et” linker isalso referred to herein as “ADL15” or an “ADL15” linker.

In some other embodiments, the Mal-spacer unit attaches the antibody orantigen binding fragment to the cleavable moiety in the linker. In someembodiments, the cleavable moiety in the linker is a cleavable peptidemoiety, e.g., an amino acid unit. In some embodiments, the cleavablepeptide moiety is Val-Cit or Val-Ala. In some embodiments, theMal-spacer unit or linker comprises MC. In some embodiments, the linkercomprises MC-Val-Cit. In some embodiments, the linker comprisesMC-Val-Ala. In some embodiments, the linker comprises MC-Glu-Val-Cit. Insome embodiments, the linker comprises MC-Ala-Ala-Asn.

In some embodiments, a spacer unit attaches the cleavable moiety in thelinker to the splicing modulator. In some embodiments, the spacer unitthat attaches the cleavable moiety to the splicing modulator isself-immolative.

In some embodiments, the spacer unit comprises pABC. In someembodiments, the pABC attaches the cleavable moiety to the splicingmodulator. In some embodiments, the cleavable moiety is a cleavablepeptide moiety, e.g., an amino acid unit. In some embodiments, thelinker comprises amino acid unit-pABC.

In some embodiments, the linker comprises Val-Cit-pABC. In someembodiments, the linker comprises Val-Cit-pABC and a MC Mal-spacer unitjoining the linker to the antibody or antigen binding fragment. In someembodiments, the linker comprises MC-Val-Cit-pABC. In some embodiments,the linker comprises MC-Val-Cit-pABC and at least one additional spacerunit. An example of an MC-Val-Cit-pABC linker is also referred to hereinas “ADL1” or an “ADL1” linker.

In some embodiments, the linker comprises Val-Ala-pABC. In someembodiments, the linker comprises Val-Ala-pABC and a MC Mal-spacer unitjoining the linker to the antibody or antigen binding fragment. In someembodiments, the linker comprises MC-Val-Ala-pABC. In some embodiments,the linker comprises MC-Val-Ala-pABC and at least one additional spacerunit. An example of an MC-Val-Ala-pABC linker is also referred to hereinas “ADL6” or an “ADL6” linker.

In some embodiments, the linker comprises Glu-Val-Cit-pABC. In someembodiments, the linker comprises Glu-Val-Cit-pABC and a MC Mal-spacerunit joining the linker to the antibody or antigen binding fragment. Insome embodiments, the linker comprises MC-Glu-Val-Cit-pABC. In someembodiments, the linker comprises MC-Glu-Val-Cit-pABC and at least oneadditional spacer unit. An example of an MC-Glu-Val-Cit-pABC linker isalso referred to herein as “ADL23” or an “ADL23” linker.

In some embodiments, the linker comprises Ala-Ala-Asn-pABC. In someembodiments, the linker comprises Ala-Ala-Asn-pABC and a MC Mal-spacerunit joining the linker to the antibody or antigen binding fragment. Insome embodiments, the linker comprises MC-Ala-Ala-Asn-pABC. In someembodiments, the linker comprises MC-Ala-Ala-Asn-pABC and at least oneadditional spacer unit. An example of an MC-Ala-Ala-Asn-pABC linker isalso referred to herein as “ADL21” or an “ADL21” linker.

In some other embodiments, the spacer unit comprises pAB. In someembodiments, the pAB attaches the cleavable moiety to the splicingmodulator. In some embodiments, the cleavable moiety is a cleavablepeptide moiety, e.g., an amino acid unit. In some embodiments, thelinker comprises amino acid unit-pAB.

In some embodiments, the linker comprises Val-Ala-pAB. In someembodiments, the linker comprises Val-Ala-pAB and an MC Mal-spacer unitjoining the linker to the antibody or antigen binding fragment. In someembodiments, the linker comprises MC-Val-Ala-pAB. In some embodiments,the linker comprises MC-Val-Ala-pAB and at least one additional spacerunit. An example of an MC-Val-Ala-pAB linker is also referred to hereinas “ADL5” or an “ADL5” linker.

In some embodiments, the linker comprises Val-Cit-pAB. In someembodiments, the linker comprises Val-Cit-pAB and an MC Mal-spacer unitjoining the linker to the antibody or antigen binding fragment. In someembodiments, the linker comprises MC-Val-Cit-pAB. In some embodiments,the linker comprises MC-Val-Cit-pAB and at least one additional spacerunit. An example of an MC-Val-Cit-pAB linker is also referred to hereinas “ADL7” or an “ADL7” linker.

In some embodiments, the linker comprises β-glucuronide-pABC. In someembodiments, the linker comprises β-glucuronide-pABC and an MCMal-spacer unit joining the linker to the antibody or antigen bindingfragment. In some embodiments, the linker comprisesMC-β-glucuronide-pABC. In some embodiments, the linker comprisesMC-β-glucuronide-pABC and at least one additional spacer unit. Anexample of an MC-β-glucuronide-pABC is also referred to herein as“ADL13” or an “ADL13” linker.

In some embodiments, the linker comprises β-glucuronide-pAB. In someembodiments, the linker comprises β-glucuronide-pAB and an MC Mal-spacerunit joining the linker to the antibody or antigen binding fragment. Insome embodiments, the linker comprises MC-β-glucuronide-pAB.

In some embodiments, the antibody or antigen binding fragment isconjugated to the splicing modulator drug moiety via an ADL1, ADL2,ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker. Ithas been discovered, in various embodiments, that ADCs comprising anADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, orADL15 linker and a splicing modulator drug moiety disclosed hereindemonstrate desirable properties for a therapeutic ADC. In variousembodiments, these properties include, but are not limited to, effectivelevels of drug loading, low aggregation levels, stability under storageconditions or when in circulation in the body (e.g., serum stability),retained affinity for target-expressing cells comparable to unconjugatedantibody, potent cytotoxicity against target-expressing cells, lowlevels of off-target cell killing, high levels of bystander killing,and/or effective in vivo anti-cancer activity, all as compared to ADCsusing other linker-payloads. For instance, in various embodiments, ADCscomprising an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21,ADL23, or ADL15 linker and a splicing modulator drug moiety disclosedherein exhibit an increased ability to inhibit growth and/orproliferation in target-expressing cells, as compared to ADCs usingother linker-payloads (e.g., an ADL10 linker and a splicing modulatordrug moiety). In various embodiments, ADCs comprising an ADL1, ADL2,ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker anda splicing modulator drug moiety disclosed herein exhibit surprisinglyincreased in vivo stability (e.g., plasma stability), as compared toother splicing modulator-based ADCs (e.g., a thailanstatin A-based ADC,for example, as reported in Puthenveetil et al. Bioconjugate Chem.(2016) 27:1880-8).

In some embodiments, the good or superior functional properties providedby the particular combination of an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12,ADL13, ADL14, ADL21, ADL23, or ADL15 linker and a splicing modulatordrug moiety disclosed herein may be observed with the linker-payloadconjugated to, e.g., an anti-HER2 antibody such as trastuzumab; ananti-CD138 antibody such as B-B4; or an anti-EPHA2 antibody such as 101.

In some embodiments, the ADC comprises ADL1-splicing modulator and anantibody or antigen binding fragment comprising an antibody or anantigen binding fragment thereof that retains the ability to target andinternalize in a neoplastic cell. In some embodiments, the ADC comprisesADL2-splicing modulator and an antibody or antigen binding fragmentcomprising an antibody or an antigen binding fragment thereof thatretains the ability to target and internalize in a neoplastic cell. Insome embodiments, the ADC comprises ADL5-splicing modulator and anantibody or antigen binding fragment comprising an antibody or anantigen binding fragment thereof that retains the ability to target andinternalize in a neoplastic cell. In some embodiments, the ADC comprisesADL6-splicing modulator and an antibody or antigen binding fragmentcomprising an antibody or an antigen binding fragment thereof thatretains the ability to target and internalize in a neoplastic cell. Insome embodiments, the ADC comprises ADL7-splicing modulator and anantibody or antigen binding fragment comprising an antibody or anantigen binding fragment thereof that retains the ability to target andinternalize in a neoplastic cell. In some embodiments, the ADC comprisesADL12-splicing modulator and an antibody or antigen binding fragmentcomprising an antibody or an antigen binding fragment thereof thatretains the ability to target and internalize in a neoplastic cell. Insome embodiments, the ADC comprises ADL13-splicing modulator and anantibody or antigen binding fragment comprising an antibody or anantigen binding fragment thereof that retains the ability to target andinternalize in a neoplastic cell. In some embodiments, the ADC comprisesADL14-splicing modulator and an antibody or antigen binding fragmentcomprising an antibody or an antigen binding fragment thereof thatretains the ability to target and internalize in a neoplastic cell. Insome embodiments, the ADC comprises ADL15-splicing modulator and anantibody or antigen binding fragment comprising an antibody or anantigen binding fragment thereof that retains the ability to target andinternalize in a neoplastic cell.

In some embodiments, the ADC comprises ADL1-splicing modulator and anantibody or antigen binding fragment thereof that targets aHER2-expressing neoplastic cell. In some embodiments, the ADC comprisesADL2-splicing modulator and an antibody or antigen binding fragmentthereof that targets a HER2-expressing neoplastic cell. In someembodiments, the ADC comprises ADL5-splicing modulator and an antibodyor antigen binding fragment thereof that targets a HER2-expressingneoplastic cell. In some embodiments, the ADC comprises ADL6-splicingmodulator and an antibody or antigen binding fragment thereof thattargets a HER2-expressing neoplastic cell. In some embodiments, the ADCcomprises ADL7-splicing modulator and an antibody or antigen bindingfragment thereof that targets a HER2-expressing neoplastic cell. In someembodiments, the ADC comprises ADL12-splicing modulator and an antibodyor antigen binding fragment thereof that targets a HER2-expressingneoplastic cell. In some embodiments, the ADC comprises ADL13-splicingmodulator and an antibody or antigen binding fragment thereof thattargets a HER2-expressing neoplastic cell. In some embodiments, the ADCcomprises ADL14-splicing modulator and an antibody or antigen bindingfragment thereof that targets a HER2-expressing neoplastic cell. In someembodiments, the ADC comprises ADL15-splicing modulator and an antibodyor antigen binding fragment thereof that targets a HER2-expressingneoplastic cell.

In some embodiments, the antibody or antigen binding fragment thereofthat targets a HER2-expressing neoplastic cell is an internalizingantibody or internalizing antigen binding fragment. In some embodiments,the antibody or antigen binding fragment thereof that targets aHER2-expressing neoplastic cell comprises three heavy chaincomplementarity determining regions (HCDRs) comprising amino acidsequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3(HCDR3); and three light chain complementarity determining regions(LCDRs) comprising amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ IDNO:5 (LCDR2), and SEQ ID NO:6 (LCDR3).

In some embodiments, the ADC has Formula (I):

Ab-(L-D)_(p)  (I)

wherein:

(i) Ab is an anti-HER2 antibody or antigen binding fragment thereofcomprising three heavy chain complementarity determining regions (HCDRs)comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2(HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementaritydetermining regions (LCDRs) comprising amino acid sequences of SEQ IDNO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3);

(ii) D is a splicing modulator;

(iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12,ADL13, ADL14, ADL21, ADL23, or ADL15; and

(iv) p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment thereofthat targets a HER2-expressing neoplastic cell comprises a heavy chainvariable region comprising an amino acid sequence of SEQ ID NO:19, and alight chain variable region comprising an amino acid sequence of SEQ IDNO:20. In some embodiments, the antibody or antigen binding fragmentthereof that targets a HER2-expressing neoplastic cell comprises a humanIgG1 heavy chain constant domain and a human Ig kappa light chainconstant domain. In some embodiments, the antibody is trastuzumab. Insome embodiments, p is an integer from 1 to 10, from 2 to 8, or from 4to 8. In some embodiments, p is 4. In some embodiments, p is 8.

In some embodiments, the ADC comprises ADL1-splicing modulator and anantibody or antigen binding fragment thereof that targets aCD138-expressing neoplastic cell. In some embodiments, the ADC comprisesADL2-splicing modulator and an antibody or antigen binding fragmentthereof that targets a CD138-expressing neoplastic cell. In someembodiments, the ADC comprises ADL5-splicing modulator and an antibodyor antigen binding fragment thereof that targets a CD138-expressingneoplastic cell. In some embodiments, the ADC comprises ADL6-splicingmodulator and an antibody or antigen binding fragment thereof thattargets a CD138-expressing neoplastic cell. In some embodiments, the ADCcomprises ADL7-splicing modulator and an antibody or antigen bindingfragment thereof that targets a CD138-expressing neoplastic cell. Insome embodiments, the ADC comprises ADL12-splicing modulator and anantibody or antigen binding fragment thereof that targets aCD138-expressing neoplastic cell. In some embodiments, the ADC comprisesADL13-splicing modulator and an antibody or antigen binding fragmentthereof that targets a CD138-expressing neoplastic cell. In someembodiments, the ADC comprises ADL14-splicing modulator and an antibodyor antigen binding fragment thereof that targets a CD138-expressingneoplastic cell. In some embodiments, the ADC comprises ADL15-splicingmodulator and an antibody or antigen binding fragment thereof thattargets a CD138-expressing neoplastic cell.

In some embodiments, the antibody or antigen binding fragment thereofthat targets a CD138-expressing neoplastic cell is an internalizingantibody or internalizing antigen binding fragment. In some embodiments,the antibody or antigen binding fragment thereof that targets aCD138-expressing neoplastic cell comprises three heavy chaincomplementarity determining regions (HCDRs) comprising amino acidsequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9(HCDR3); and three light chain complementarity determining regions(LCDRs) comprising amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ IDNO:11 (LCDR2), and SEQ ID NO:12 (LCDR3).

In some embodiments, the ADC has Formula (I):

Ab-(L-D)_(p)  (I)

wherein:

(i) Ab is an anti-CD138 antibody or antigen binding fragment thereofcomprising three heavy chain complementarity determining regions (HCDRs)comprising amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8(HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementaritydetermining regions (LCDRs) comprising amino acid sequences of SEQ IDNO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3);

(ii) D is a splicing modulator;

(iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12,ADL13, ADL14, ADL21, ADL23, or ADL15; and

(iv) p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment thereofthat targets a CD138-expressing neoplastic cell comprises a heavy chainvariable region comprising an amino acid sequence of SEQ ID NO:21, and alight chain variable region comprising an amino acid sequence of SEQ IDNO:22. In some embodiments, the antibody or antigen binding fragmentthereof that targets a CD138-expressing neoplastic cell comprises amurine IgG2a heavy chain constant domain and a murine Ig kappa lightchain constant domain. In some embodiments, the antibody or antigenbinding fragment thereof that targets a CD138-expressing neoplastic cellcomprises a human IgG2a heavy chain constant domain and a human Ig kappalight chain constant domain. In some embodiments, the antibody is B-B4.In some embodiments, p is an integer from 1 to 10, from 2 to 8, or from4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

In some embodiments, the ADC comprises ADL1-splicing modulator and anantibody or antigen binding fragment thereof that targets anEPHA2-expressing neoplastic cell. In some embodiments, the ADC comprisesADL2-splicing modulator and an antibody or antigen binding fragmentthereof that targets an EPHA2-expressing neoplastic cell. In someembodiments, the ADC comprises ADL5-splicing modulator and an antibodyor antigen binding fragment thereof that targets an EPHA2-expressingneoplastic cell. In some embodiments, the ADC comprises ADL6-splicingmodulator and an antibody or antigen binding fragment thereof thattargets an EPHA2-expressing neoplastic cell. In some embodiments, theADC comprises ADL7-splicing modulator and an antibody or antigen bindingfragment thereof that targets an EPHA2-expressing neoplastic cell. Insome embodiments, the ADC comprises ADL12-splicing modulator and anantibody or antigen binding fragment thereof that targets anEPHA2-expressing neoplastic cell. In some embodiments, the ADC comprisesADL13-splicing modulator and an antibody or antigen binding fragmentthereof that targets an EPHA2-expressing neoplastic cell. In someembodiments, the ADC comprises ADL14-splicing modulator and an antibodyor antigen binding fragment thereof that targets an EPHA2-expressingneoplastic cell. In some embodiments, the ADC comprises ADL15-splicingmodulator and an antibody or antigen binding fragment thereof thattargets an EPHA2-expressing neoplastic cell.

In some embodiments, the antibody or antigen binding fragment thereofthat targets an EPHA2-expressing neoplastic cell is an internalizingantibody or internalizing antigen binding fragment. In some embodiments,the antibody or antigen binding fragment thereof that targets anEPHA2-expressing neoplastic cell comprises three heavy chaincomplementarity determining regions (HCDRs) comprising amino acidsequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ IDNO:15 (HCDR3); and three light chain complementarity determining regions(LCDRs) comprising amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ IDNO:17 (LCDR2), and SEQ ID NO:18 (LCDR3).

In some embodiments, the ADC has Formula (I):

Ab-(L-D)_(p)  (I)

wherein:

(i) Ab is an anti-EPHA2 antibody or antigen binding fragment thereofcomprising three heavy chain complementarity determining regions (HCDRs)comprising amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14(HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementaritydetermining regions (LCDRs) comprising amino acid sequences of SEQ IDNO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO:18 (LCDR3);

(ii) D is a splicing modulator;

(iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12,ADL13, ADL14, ADL21, ADL23, or ADL15; and

(iv) p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment thereofthat targets an EPHA2-expressing neoplastic cell comprises a heavy chainvariable region comprising an amino acid sequence of SEQ ID NO:23, and alight chain variable region comprising an amino acid sequence of SEQ IDNO:24. In some embodiments, the antibody or antigen binding fragmentthereof that targets an EPHA2-expressing neoplastic cell comprises ahuman IgG1 heavy chain constant domain and a human Ig kappa light chainconstant domain. In some embodiments, the antibody is 101. In someembodiments, p is an integer from 1 to 10, from 2 to 8, or from 4 to 8.In some embodiments, p is 4. In some embodiments, p is 8.

Drug Moieties

The drug moiety (D) of the ADCs described herein can be anychemotherapeutic agent. Useful classes of chemotherapeutic agentsinclude, for example, modulators of RNA splicing. In certain preferredembodiments, the drug moiety is a splicing modulator. Exemplary splicingmodulator compounds are described and exemplified herein.

In various embodiments, the drug moiety is a splicing modulator compoundof Formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is chosen from absent, hydrogen, C₁-C₆ alkyl groups, C₁-C₆alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acidgroups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzylgroups, C₃-C₈ heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl) groups, and—CD₃;

R³ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxygroups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups,C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, and —O—C(═O)—(C₁-C₆ alkyl) groups; and

R⁴, R⁵, and R⁸ are each independently chosen from hydrogen, hydroxylgroups, —O—(C₁-C₆ alkyl) groups, —O—C(═O)—(C₁-C₆ alkyl) groups, andC₁-C₆ alkyl groups;

R⁶ and R⁷ are each independently chosen from hydrogen, —O—R¹⁷,—O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₆ alkyl groups, and —NR¹⁵R¹⁶;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷;

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups; and

Z is chosen from

wherein R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, R¹⁶, and R¹⁷ are eachindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups,—NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆alkylalkoxy groups, benzyl groups, and C₃-C₈ heterocyclyl groups,

wherein at least one of R⁶ and R⁷ is hydrogen.

In some embodiments, R¹ is chosen from hydrogen, C₁-C₄ alkyl groups,C₁-C₄ alkylcarboxylic acid groups, and C₃-C₈ cycloalkyl groups. In someembodiments, R¹ is hydrogen. In some embodiments, R¹ is a C₁-C₄ alkylgroup. In some embodiments, R¹ is methyl. In some embodiments, R¹ isethyl. In some embodiments, R¹ is a C₁-C₄ alkylcarboxylic acid group. Insome embodiments, R¹ is —CH₂CH₂CH₂CO₂H. In some embodiments, R¹ is aC₃-C₈ cycloalkyl group. In some embodiments, R¹ is cycloheptyl.

In some embodiments, R³ is chosen from hydrogen, C₁-C₄ alkyl groups,C₁-C₄ alkylalkoxy groups, C₁-C₄ alkylcarboxylic acid groups, and C₁-C₄alkylhydroxy groups. In some embodiments, R³ is chosen from hydrogen andC₁-C₄ alkylcarboxylic acid groups. In some embodiments, R³ is hydrogen.In some embodiments, R³ is a C₁-C₄ alkylcarboxylic acid group. In someembodiments, R³ is —CH₂CH₂CO₂H.

In some embodiments, R⁴ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, —O—C(═O)—(C₁-C₄ alkyl) groups, and C₁-C₄ alkylgroups. In some embodiments, R⁴ is hydrogen. In some embodiments, R⁴ ishydroxyl. In some embodiments, R⁴ is a —O—(C₁-C₄ alkyl) group. In someembodiments, R⁴ is —OCH₃. In some embodiments, R⁴ is —OCH₂CH₃. In someembodiments, R⁴ is a —O—C(═O)—(C₁-C₄ alkyl) group. In some embodiments,R⁴ is —O—C(═O)—CH₃. In some embodiments, R⁴ is —O—C(═O)—CH₂CH₃. In someembodiments, R⁴ is a C₁-C₄ alkyl group. In some embodiments, R⁴ ismethyl. In some embodiments, R⁴ is ethyl.

In some embodiments, R⁵ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, and C₁-C₄ alkyl groups. In some embodiments, R⁵is hydrogen. In some embodiments, R⁵ is hydroxyl. In some embodiments,R⁵ is a —O—(C₁-C₄ alkyl) group. In some embodiments, R⁵ is a C₁-C₄ alkylgroup.

In some embodiments, R⁶ is hydrogen. In some embodiments, R⁷ ishydrogen. In some embodiments, R⁶ is hydrogen and R⁷ is —O—R¹⁷. In someembodiments, R⁶ is hydrogen and R⁷ is —OR¹⁷, wherein R¹⁷ is chosen fromhydrogen and C¹-C⁴ alkyl groups. In some embodiments, R⁶ is hydrogen andR⁷ is —O—R¹⁷, wherein R¹⁷ is hydrogen. In some embodiments, R⁶ is —O—R¹⁷and R⁷ is hydrogen. In some embodiments, R⁶ is —O—R¹⁷ and R⁷ ishydrogen, wherein R¹⁷ is chosen from hydrogen and C¹-C⁴ alkyl groups. Insome embodiments, R⁶ is —O—R¹⁷ and R⁷ is hydrogen, wherein R¹⁷ ishydrogen. In some embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶. Insome embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶, wherein R¹⁵ is Hand R¹⁶ is chosen from hydrogen, R¹⁷, —C(═O)—R¹⁷, and —C(═O)—O—R¹⁷. Insome embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶, wherein R¹⁵ is Hand R¹⁶ is chosen from hydrogen, R¹⁷, —C(═O)—R¹⁷, and —C(═O)—O—R¹⁷, andwherein R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In some embodiments,R⁶ is —O—R¹⁷. In some embodiments, R⁶ is —O—C(═O)—R¹⁷. In someembodiments, R⁶ is C₁-C₆ alkyl. In some embodiments, R⁶ is C₁-C₄ alkyl.In some embodiments, R⁶ is C₁ alkyl. In some embodiments, R⁶ is—NR¹⁵R¹⁶. In some embodiments, R⁷ is —O—R¹⁷. In some embodiments, R⁷ is—O—C(═O)—R¹⁷. In some embodiments, R⁷ is C₁-C₆ alkyl. In someembodiments, R⁷ is C₁-C₄ alkyl. In some embodiments, R⁷ is C₁ alkyl. Insome embodiments, R⁷ is —NR¹⁵R¹⁶.

In some embodiments, R⁸ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, and (C₁-C₄ alkyl). In some embodiments, R⁸ ishydrogen. In some embodiments, R⁸ is a hydroxyl group. In someembodiments, R⁸ is an —O—(C₁-C₄ alkyl) group. In some embodiments, R⁸ isan —O—(C₁ alkyl) group.

In some embodiments, R¹⁵ is hydrogen. In some embodiments, R¹⁵ is R¹⁷.In some embodiments, R¹⁵ is —C(═O)—R¹⁷. In some embodiments, R¹⁵ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁶ is hydrogen. In some embodiments, R¹⁶ is R¹⁷.In some embodiments, R¹⁶ is —C(═O)—R¹⁷. In some embodiments, R¹⁶ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁷ is chosen from hydrogen, C₁-C₄ alkyl groups,C₃-C₆ cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In someembodiments, R¹⁷ is hydrogen. In some embodiments, R¹⁷ is a C₁-C₄ alkylgroup. In some embodiments, R¹⁷ is a C₁ alkyl group. In someembodiments, R¹⁷ is a C₃-C₆ cycloalkyl group. In some embodiments, R¹⁷is a C₃ cycloalkyl group. In some embodiments, R¹⁷ is a C₄ cycloalkylgroup. In some embodiments, R¹⁷ is a C₅ cycloalkyl group. In someembodiments, R¹⁷ is a C₆ cycloalkyl group. In some embodiments, R¹⁷ is aC₃-C₈ heterocyclyl group. In some embodiments, R¹⁷ is a C₃ heterocyclylgroup. In some embodiments, R¹⁷ is a C₄ heterocyclyl group. In someembodiments, R¹⁷ is a C₅ heterocyclyl group. In some embodiments, R¹⁷ isa C₆ heterocyclyl group. In some embodiments, R¹⁷ is a C₇ heterocyclylgroup. In some embodiments, R¹⁷ is a C₈ heterocyclyl group.

In some embodiments, Z is In some embodiments, Z is

In some embodiments, Z is In some embodiments, Z is

In some embodiments, Z is

In some embodiments, the splicing modulator compound of Formula (II)attaches to the linker L, e.g., in an ADC of Formula (I), as shown inFormula (II-A):

wherein Z′ is chosen from and

wherein all other variables are as defined for Formula (II).

In various other embodiments, the drug moiety is a splicing modulatorcompound of Formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxygroups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups,C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl) groups, and —CD₃;

R³ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxygroups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups,C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, and —O—C(═O)—(C₁-C₆ alkyl) groups; and

R⁴, R⁵, and R⁸ are each independently chosen from hydrogen, hydroxylgroups, —O—(C₁-C₆ alkyl) groups, —O—C(═O)—(C₁-C₆ alkyl) groups, andC₁-C₆ alkyl groups;

R⁶ and R⁷ are each independently chosen from hydrogen, —O—R¹⁷,—O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₆ alkyl groups, and —NR¹⁵R¹⁶;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷; and

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, R¹⁶, and R¹⁷ are eachindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups,—NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆alkylalkoxy groups, benzyl groups, and C₃-C₈ heterocyclyl groups,wherein at least one of R⁶ and R⁷ is hydrogen.

In some embodiments, R¹ is chosen from hydrogen, C₁-C₄ alkyl groups,C₁-C₄ alkylcarboxylic acid groups, and C₃-C₈ cycloalkyl groups. In someembodiments, R¹ is hydrogen. In some embodiments, R¹ is a C₁-C₄ alkylgroup. In some embodiments, R¹ is methyl. In some embodiments, R¹ isethyl. In some embodiments, R¹ is a C₁-C₄ alkylcarboxylic acid group. Insome embodiments, R¹ is —CH₂CH₂CH₂CO₂H. In some embodiments, R¹ is aC₃-C₈ cycloalkyl group. In some embodiments, R¹ is cycloheptyl.

In some embodiments, R³ is chosen from hydrogen, C₁-C₄ alkyl groups,C₁-C₄ alkylalkoxy groups, C₁-C₄ alkylcarboxylic acid groups, and C₁-C₄alkylhydroxy groups. In some embodiments, R³ is chosen from hydrogen andC₁-C₄ alkylcarboxylic acid groups. In some embodiments, R³ is hydrogen.In some embodiments, R³ is a C₁-C₄ alkylcarboxylic acid group. In someembodiments, R³ is —CH₂CH₂CO₂H.

In some embodiments, R⁴ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, —O—C(═O)—(C₁-C₄ alkyl) groups, and C₁-C₄ alkylgroups. In some embodiments, R⁴ is hydrogen. In some embodiments, R⁴ ishydroxyl. In some embodiments, R⁴ is a —O—(C₁-C₄ alkyl) group. In someembodiments, R⁴ is —OCH₃. In some embodiments, R⁴ is —OCH₂CH₃. In someembodiments, R⁴ is a —O—C(═O)—(C₁-C₄ alkyl) group. In some embodiments,R⁴ is —O—C(═O)—CH₃. In some embodiments, R⁴ is —O—C(═O)—CH₂CH₃. In someembodiments, R⁴ is a C₁-C₄ alkyl group. In some embodiments, R⁴ ismethyl. In some embodiments, R⁴ is ethyl.

In some embodiments, R⁵ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, and C₁-C₄ alkyl groups. In some embodiments, R⁵is hydrogen. In some embodiments, R⁵ is hydroxyl. In some embodiments,R⁵ is a —O—(C₁-C₄ alkyl) group. In some embodiments, R⁵ is a C₁-C₄ alkylgroup.

In some embodiments, R⁶ is hydrogen. In some embodiments, R⁷ ishydrogen. In some embodiments, R⁶ is hydrogen and R⁷ is —O—R¹⁷. In someembodiments, R⁶ is hydrogen and R⁷ is —OR¹⁷, wherein R¹⁷ is chosen fromhydrogen and C¹-C⁴ alkyl groups. In some embodiments, R⁶ is hydrogen andR⁷ is —O—R¹⁷, wherein R¹⁷ is hydrogen. In some embodiments, R⁶ is —O—R¹⁷and R⁷ is hydrogen. In some embodiments, R⁶ is —O—R¹⁷ and R⁷ ishydrogen, wherein R¹⁷ is chosen from hydrogen and C¹-C⁴ alkyl groups. Insome embodiments, R⁶ is —O—R¹⁷ and R⁷ is hydrogen, wherein R¹⁷ ishydrogen. In some embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶. Insome embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶, wherein R¹⁵ is Hand R¹⁶ is chosen from hydrogen, R¹⁷, —C(═O)—R¹⁷, and —C(═O)—O—R¹⁷. Insome embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶, wherein R¹⁵ is Hand R¹⁶ is chosen from hydrogen, R¹⁷, —C(═O)—R¹⁷, and —C(═O)—O—R¹⁷, andwherein R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In some embodiments,R⁶ is —O—R¹⁷. In some embodiments, R⁶ is —O—C(═O)—R¹⁷. In someembodiments, R⁶ is C₁-C₆ alkyl. In some embodiments, R⁶ is C₁-C₄ alkyl.In some embodiments, R⁶ is C₁ alkyl. In some embodiments, R⁶ is—NR¹⁵R¹⁶.

In some embodiments, R⁷ is —O—R¹⁷. In some embodiments, R⁷ is—O—C(═O)—R¹⁷. In some embodiments, R⁷ is C₁-C₆ alkyl. In someembodiments, R⁷ is C₁-C₄ alkyl. In some embodiments, R⁷ is C₁ alkyl. Insome embodiments, R⁷ is —NR¹⁵R¹⁶.

In some embodiments, R⁸ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, and (C₁-C₄ alkyl). In some embodiments, R⁸ ishydrogen. In some embodiments, R⁸ is a hydroxyl group. In someembodiments, R⁸ is an —O—(C₁-C₄ alkyl) group. In some embodiments, R⁸ isan —O—(C₁ alkyl) group.

In some embodiments, R¹⁵ is hydrogen. In some embodiments, R¹⁵ is R¹⁷.In some embodiments, R¹⁵ is —C(═O)—R¹⁷. In some embodiments, R¹⁵ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁶ is hydrogen. In some embodiments, R¹⁶ is R¹⁷.In some embodiments, R¹⁶ is —C(═O)—R¹⁷. In some embodiments, R¹⁶ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁷ is chosen from hydrogen, C₁-C₄ alkyl groups,C₃-C₆ cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In someembodiments, R¹⁷ is hydrogen. In some embodiments, R¹⁷ is a C₁-C₄ alkylgroup. In some embodiments, R¹⁷ is a C₁ alkyl group. In someembodiments, R¹⁷ is a C₃-C₆ cycloalkyl group. In some embodiments, R¹⁷is a C₃ cycloalkyl group. In some embodiments, R¹⁷ is a C₄ cycloalkylgroup. In some embodiments, R¹⁷ is a C₅ cycloalkyl group. In someembodiments, R¹⁷ is a C₆ cycloalkyl group. In some embodiments, R¹⁷ is aC₃-C₈ heterocyclyl group. In some embodiments, R¹⁷ is a C₃ heterocyclylgroup. In some embodiments, R¹⁷ is a C₄ heterocyclyl group. In someembodiments, R¹⁷ is a C₅ heterocyclyl group. In some embodiments, R¹⁷ isa C₆ heterocyclyl group. In some embodiments, R¹⁷ is a C₇ heterocyclylgroup. In some embodiments, R¹⁷ is a C₈ heterocyclyl group.

In some embodiments, the splicing modulator compound of Formula (IV)attaches to the linker L, e.g., in an ADC of Formula (I), as shown inFormula (IV-A):

In various other embodiments, the drug moiety is a splicing modulatorcompound of Formula (VI):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R⁹ are each independently chosen from hydrogen, C₁-C₆ alkylgroups, C₁-C₆ alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆alkylcarboxylic acid groups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkylgroups, benzyl groups, C₃-C₈ heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl)groups, and —CD₃;

R³ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxygroups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups,C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, and —O—C(═O)—(C₁-C₆ alkyl) groups;

R⁴, R⁵, and R⁸ are each independently chosen from hydrogen, hydroxylgroups, —O—(C₁-C₆ alkyl) groups, —O—C(═O)—(C₁-C₆ alkyl) groups, andC₁-C₆ alkyl groups;

R⁶ and R⁷ are each independently chosen from hydrogen, —O—R¹⁷,—O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₆ alkyl groups, —NR¹⁵R¹⁶, and alinker;

R¹⁰ is chosen from hydrogen, C₁-C₆ alkyl groups, —C(═O)—(C₁-C₆ alkyl)groups, and —CD₃;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷;

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups; and

a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

wherein R¹, R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹⁵, R¹⁶, and R¹⁷ are eachindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups,—NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆alkylalkoxy groups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein at least one of R⁶ and R⁷ is hydrogen; and

wherein R¹ and R⁹ cannot both be absent.

In some embodiments, R¹ is chosen from hydrogen, C₁-C₄ alkyl groups,C₁-C₄ alkylcarboxylic acid groups, and C₃-C₈ cycloalkyl groups. In someembodiments, R¹ is hydrogen. In some embodiments, R¹ is a C₁-C₄ alkylgroup. In some embodiments, R¹ is methyl. In some embodiments, R¹ isethyl. In some embodiments, R¹ is a C₁-C₄ alkylcarboxylic acid group. Insome embodiments, R¹ is —CH₂CH₂CH₂CO₂H. In some embodiments, R¹ is aC₃-C₈ cycloalkyl group. In some embodiments, R¹ is cycloheptyl.

In some embodiments, R³ is chosen from hydrogen, C₁-C₄ alkyl groups,C₁-C₄ alkylalkoxy groups, C₁-C₄ alkylcarboxylic acid groups, and C₁-C₄alkylhydroxy groups. In some embodiments, R³ is chosen from hydrogen andC₁-C₄ alkylcarboxylic acid groups. In some embodiments, R³ is hydrogen.In some embodiments, R³ is a C₁-C₄ alkylcarboxylic acid group. In someembodiments, R³ is —CH₂CH₂CO₂H.

In some embodiments, R⁴ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, —O—C(═O)—(C₁-C₄ alkyl) groups, and C₁-C₄ alkylgroups. In some embodiments, R⁴ is hydrogen. In some embodiments, R⁴ ishydroxyl. In some embodiments, R⁴ is a —O—(C₁-C₄ alkyl) group. In someembodiments, R⁴ is —OCH₃. In some embodiments, R⁴ is —OCH₂CH₃. In someembodiments, R⁴ is a —O—C(═O)—(C₁-C₄ alkyl) group. In some embodiments,R⁴ is —O—C(═O)—CH₃. In some embodiments, R⁴ is —O—C(═O)—CH₂CH₃. In someembodiments, R⁴ is a C₁-C₄ alkyl group. In some embodiments, R⁴ ismethyl. In some embodiments, R⁴ is ethyl.

In some embodiments, R⁵ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, and C₁-C₄ alkyl groups. In some embodiments, R⁵is hydrogen. In some embodiments, R⁵ is hydroxyl. In some embodiments,R⁵ is a —O—(C₁-C₄ alkyl) group. In some embodiments, R⁵ is a C₁-C₄ alkylgroup.

In some embodiments, R⁹ is chosen from absent, hydrogen, C₁-C₄ alkylgroups, —(C═O)—(C₁-C₄ alkyl) groups, and —CD₃. In some embodiments, R⁹is absent. In some embodiments, R⁹ is hydrogen. In some embodiments, R⁹is a C₁-C₄ alkyl group. In some embodiments, the C₁-C₄ alkyl group ismethyl. In some embodiments, the C₁-C₄ alkyl group is ethyl. In someembodiments, R⁹ is a —(C═O)—(C₁-C₄ alkyl) group. In some embodiments,the —(C═O)—(C₁-C₄ alkyl) group is —(C═O)-methyl. In some embodiments, R⁹is —CD₃.

In some embodiments, R¹⁰ is chosen from hydrogen, C₁-C₄ alkyl groups,—(C═O)—(C₁-C₄ alkyl) groups, and —CD₃. In some embodiments, R¹⁰ ishydrogen. In some embodiments, R¹⁰ is a C₁-C₄ alkyl group. In someembodiments, the C₁-C₄ alkyl group is methyl. In some embodiments, theC₁-C₄ alkyl group is ethyl. In some embodiments, R¹⁰ is a —(C═O)—(C₁-C₄alkyl) group. In some embodiments, the —(C═O)—(C₁-C₄ alkyl) group is—(C═O)-methyl. In some embodiments, R¹⁰ is —CD₃.

In some embodiments, R⁶ is hydrogen. In some embodiments, R⁷ ishydrogen. In some embodiments, R⁶ is hydrogen and R⁷ is —O—R¹⁷. In someembodiments, R⁶ is hydrogen and R⁷ is —OR¹⁷, wherein R¹⁷ is chosen fromhydrogen and C¹-C⁴ alkyl groups. In some embodiments, R⁶ is hydrogen andR⁷ is —O—R¹⁷, wherein R¹⁷ is hydrogen. In some embodiments, R⁶ is —O—R¹⁷and R⁷ is hydrogen. In some embodiments, R⁶ is —O—R¹⁷ and R⁷ ishydrogen, wherein R¹⁷ is chosen from hydrogen and C¹-C⁴ alkyl groups. Insome embodiments, R⁶ is —O—R¹⁷ and R⁷ is hydrogen, wherein R¹⁷ ishydrogen. In some embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶. Insome embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶, wherein R¹⁵ is Hand R¹⁶ is chosen from hydrogen, R¹⁷, —C(═O)—R¹⁷, and —C(═O)—O—R¹⁷. Insome embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶, wherein R¹⁵ is Hand R¹⁶ is chosen from hydrogen, R¹⁷, —C(═O)—R¹⁷, and —C(═O)—O—R¹⁷, andwherein R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In some embodiments,R⁶ is —O—R¹⁷. In some embodiments, R⁶ is —O—C(═O)—R¹⁷. In someembodiments, R⁶ is C₁-C₆ alkyl. In some embodiments, R⁶ is C₁-C₄ alkyl.In some embodiments, R⁶ is C₁ alkyl. In some embodiments, R⁶ is—NR¹⁵R¹⁶.

In some embodiments, R⁷ is —O—R¹⁷. In some embodiments, R⁷ is—O—C(═O)—R¹⁷. In some embodiments, R⁷ is C₁-C₆ alkyl. In someembodiments, R⁷ is C₁-C₄ alkyl. In some embodiments, R⁷ is C₁ alkyl. Insome embodiments, R⁷ is —NR¹⁵R¹⁶.

In some embodiments, R⁸ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, and (C₁-C₄ alkyl). In some embodiments, R⁸ ishydrogen. In some embodiments, R⁸ is a hydroxyl group. In someembodiments, R⁸ is an —O—(C₁-C₄ alkyl) group. In some embodiments, R⁸ isan —O—(C₁ alkyl) group.

In some embodiments, R¹⁵ is hydrogen. In some embodiments, R¹⁵ is R¹⁷.In some embodiments, R¹⁵ is —C(═O)—R¹⁷. In some embodiments, R¹⁵ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁶ is hydrogen. In some embodiments, R¹⁶ is R¹⁷.In some embodiments, R¹⁶ is —C(═O)—R¹⁷. In some embodiments, R¹⁶ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁷ is chosen from hydrogen, C₁-C₄ alkyl groups,C₃-C₆ cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In someembodiments, R¹⁷ is hydrogen. In some embodiments, R¹⁷ is a C₁-C₄ alkylgroup. In some embodiments, R¹⁷ is a C₁ alkyl group. In someembodiments, R¹⁷ is a C₃-C₆ cycloalkyl group. In some embodiments, R¹⁷is a C₃ cycloalkyl group. In some embodiments, R¹⁷ is a C₄ cycloalkylgroup. In some embodiments, R¹⁷ is a C₅ cycloalkyl group. In someembodiments, R¹⁷ is a C₆ cycloalkyl group. In some embodiments, R¹⁷ is aC₃-C₈ heterocyclyl group. In some embodiments, R¹⁷ is a C₃ heterocyclylgroup. In some embodiments, R¹⁷ is a C₄ heterocyclyl group. In someembodiments, R¹⁷ is a C₅ heterocyclyl group. In some embodiments, R¹⁷ isa C₆ heterocyclyl group. In some embodiments, R¹⁷ is a C₇ heterocyclylgroup. In some embodiments, R¹⁷ is a C₈ heterocyclyl group.

In some embodiments, a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, a is 1, 2, 3, 4, 5, or 6. In some embodiments, a is 1, 2,3, 4, or 5. In some embodiments, a is 1, 2, 3, or 4. In someembodiments, a is 1, 2, or 3. In some embodiments, a is 1 or 2. In someembodiments, a is 1. In some embodiments, a is 2. In some embodiments, ais 3. In some embodiments, a is 4. In some embodiments, a is 5. In someembodiments, a is 6. In some embodiments, a is 7. In some embodiments, ais 8. In some embodiments, a is 9. In some embodiments, a is 10.

In some embodiments, the splicing modulator compound of Formula (VI)attaches to the linker L, e.g., in an ADC of Formula (I), as shown inFormula (VI-A):

In various other embodiments, the drug moiety is a splicing modulatorcompound of Formula (VIII):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is chosen from absent, hydrogen, C₁-C₆ alkyl groups, C₁-C₆alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acidgroups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzylgroups, C₃-C₈ heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl) groups, and—CD₃;

R³ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxygroups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups,C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, and —O—C(═O)—(C₁-C₆ alkyl) groups;

R⁴ is chosen from hydrogen, hydroxyl groups, —O—(C₁-C₆ alkyl) groups,—O—C(═O)—(C₁-C₆ alkyl) groups, and C₁-C₆ alkyl groups; and

R¹⁰ is chosen from 3 to 10 membered carbocycles and 3 to 10 memberedheterocycles, each of which is substituted with 0 to 3 R^(a), whereineach R^(a) is independently chosen from halogens, C₁-C₆ alkyl groups,—O—(C₁-C₆)alkyl groups, C₁-C₆ alkylalkoxy groups, C₁-C₆ alkylhydroxygroups, —S(═O)_(w)-(4 to 7 membered heterocycles), 4 to 7 memberedcarbocycles, and 4 to 7 membered heterocycles;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷; and

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein R¹, R³, R⁴, R¹⁰, R¹⁵, R¹⁶, and R¹⁷ are each independentlysubstituted with 0 to 3 groups independently chosen from halogens,hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups, —NR¹⁵R¹⁶,C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆ alkylalkoxygroups, benzyl groups, and C₃-C₈ heterocyclyl groups; and wherein eachR^(a) is independently substituted with 0 to 3 groups independentlychosen from halogens, hydroxyl groups, —NR¹⁵R¹⁶, C₁-C₆ alkyl groups,—(C═O)—(C₁-C₆ alkyl) groups, —(C═O)—(C₁-C₆ alkyl)-(C₃-C₁₀ heterocyclylgroups), and C₁-C₆ alkylcarboxylic acid groups, each of which issubstituted with 0, 1, or 2 groups independently chosen from halogens,hydroxyl groups, —NR¹⁵R¹⁶, and C₁-C₃ alkyl groups; and

w is 0, 1, or 2.

In some embodiments, R¹ is chosen from absent, hydrogen, C₁-C₄ alkylgroups, C₁-C₄ alkylcarboxylic acid groups, and C₃-C₈ cycloalkyl groups.In some embodiments, R¹ is hydrogen. In some embodiments, R¹ is a C₁-C₄alkyl group. In some embodiments, R¹ is methyl. In some embodiments, R¹is ethyl. In some embodiments, R¹ is a C₁-C₄ alkylcarboxylic acid group.In some embodiments, R¹ is —CH₂CH₂CH₂CO₂H. In some embodiments, R¹ is aC₃-C₈ cycloalkyl group. In some embodiments, R¹ is cycloheptyl.

In some embodiments, R³ is chosen from hydrogen, C₁-C₄ alkyl groups,C₁-C₄ alkylalkoxy groups, C₁-C₄ alkylcarboxylic acid groups, and C₁-C₄alkylhydroxy groups. In some embodiments, R³ is chosen from hydrogen andC₁-C₄ alkylcarboxylic acid groups. In some embodiments, R³ is hydrogen.In some embodiments, R³ is a C₁-C₄ alkylcarboxylic acid group. In someembodiments, R³ is —CH₂CH₂CO₂H.

In some embodiments, R⁴ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, —O—C(═O)—(C₁-C₄ alkyl) groups, and C₁-C₄ alkylgroups. In some embodiments, R⁴ is hydrogen. In some embodiments, R⁴ ishydroxyl. In some embodiments, R⁴ is a —O—(C₁-C₄ alkyl) group. In someembodiments, R⁴ is —OCH₃. In some embodiments, R⁴ is —OCH₂CH₃. In someembodiments, R⁴ is a —O—C(═O)—(C₁-C₄ alkyl) group. In some embodiments,R⁴ is —O—C(═O)—CH₃. In some embodiments, R⁴ is —O—C(═O)—CH₂CH₃. In someembodiments, R⁴ is a C₁-C₄ alkyl group. In some embodiments, R⁴ ismethyl. In some embodiments, R⁴ is ethyl.

In some embodiments, R¹⁰ is chosen from 6 to 9 membered carbocycles and6 to 9 membered heterocycles, each of which is substituted with 0 to 2R^(a), wherein each R^(a) is independently substituted with 0 to 3groups independently chosen from halogens, hydroxyl groups, C₁-C₆ alkylgroups, —(C═O)—(C₁-C₆ alkyl) groups, —(C═O)—(C₁-C₆ alkyl)-(3 to 10membered heterocycle) groups, and C₁-C₆ alkylcarboxylic acid groups.

In some embodiments, the carbocycle is a phenyl substituted with 0 to 2R^(a), wherein each R^(a) is independently substituted with 0 to 3groups independently chosen from halogens, hydroxyl groups, C₁-C₆ alkylgroups, —(C═O)—(C₁-C₆ alkyl) groups, —(C═O)—(C₁-C₆ alkyl)-(3 to 10membered heterocycle) groups, and C₁-C₆ alkylcarboxylic acid groups. Insome embodiments, the phenyl is substituted with 2 R^(a), wherein eachR^(a) is independently substituted with 0 to 3 groups independentlychosen from halogens, hydroxyl groups, C₁-C₆ alkyl groups, —(C═O)—(C₁-C₆alkyl) groups, —(C═O)—(C₁-C₆ alkyl)-(3 to 10 membered heterocycle)groups, and C₁-C₆ alkylcarboxylic acid groups. In some embodiments, thephenyl is

In some embodiments, the heterocycle is a 9 membered heterocyclesubstituted with 0 to 2 R^(a), wherein each R^(a) is independentlysubstituted with 0 to 3 groups independently chosen from halogens,hydroxyl groups, C₁-C₆ alkyl groups, —(C═O)—(C₁-C₆ alkyl) groups,—(C═O)—(C₁-C₆ alkyl)-(3 to 10 membered heterocycle) groups, and C₁-C₆alkylcarboxylic acid groups. In some embodiments, the 9 memberedheterocycle is

In some embodiments, R^(a) is chosen from halogens, 3 to 10 memberedcarbocycles, and 3 to 10 membered heterocycles, wherein each R^(a) isindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups, —(C═O)—(C₁-C₆ alkyl)groups, —(C═O)—(C₁-C₆ alkyl)-(3 to 10 membered heterocycle) groups, andC₁-C₆ alkylcarboxylic acid groups. In some embodiments, R^(a) is chosenfrom halogens,

In some embodiments, R¹⁵ is hydrogen. In some embodiments, R¹⁵ is R¹⁷.In some embodiments, R¹⁵ is —C(═O)—R¹⁷. In some embodiments, R¹⁵ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁶ is hydrogen. In some embodiments, R¹⁶ is R¹⁷.In some embodiments, R¹⁶ is —C(═O)—R¹⁷. In some embodiments, R¹⁶ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁷ is chosen from hydrogen, C₁-C₄ alkyl groups,C₃-C₆ cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In someembodiments, R¹⁷ is hydrogen. In some embodiments, R¹⁷ is a C₁-C₄ alkylgroup. In some embodiments, R¹⁷ is a C₁ alkyl group. In someembodiments, R¹⁷ is a C₃-C₆ cycloalkyl group. In some embodiments, R¹⁷is a C₃ cycloalkyl group. In some embodiments, R¹⁷ is a C₄ cycloalkylgroup. In some embodiments, R¹⁷ is a C₅ cycloalkyl group. In someembodiments, R¹⁷ is a C₆ cycloalkyl group. In some embodiments, R¹⁷ is aC₃-C₈ heterocyclyl group. In some embodiments, R¹⁷ is a C₃ heterocyclylgroup. In some embodiments, R¹⁷ is a C₄ heterocyclyl group. In someembodiments, R¹⁷ is a C₅ heterocyclyl group. In some embodiments, R¹⁷ isa C₆ heterocyclyl group. In some embodiments, R¹⁷ is a C₇ heterocyclylgroup. In some embodiments, R¹⁷ is a C₈ heterocyclyl group.

In some embodiments, the splicing modulator compound of Formula (VIII)attaches to the linker L, e.g., in an ADC of Formula (I), as shown inFormula (VIII-A):

In various embodiments, the drug moiety is a splicing modulator selectedfrom D2 and D1.

In various embodiments, the drug moiety is D2. In various embodiments,the structure of the D2 drug moiety used in the disclosed ADCs is shownbelow:

In various embodiments, the linker in the ADCs (e.g., ADCs of Formula(I)) described herein covalently attaches to the D2 drug moiety via anamine on the piperazine group. In various embodiments, the drug moietyis a derivative of D2. In various embodiments, the D2 derivative retainsat least one biological function or activity as D2 (e.g., SF3b complexbinding, in vitro splicing activity, cytotoxicity) but has an alteredchemical structure.

In various embodiments, the drug moiety is D1 or a pharmaceuticallyacceptable salt thereof. In various embodiments, the structure of the D1drug moiety used in the disclosed ADCs is shown below:

In various embodiments, the linker in the ADCs (e.g., ADCs of Formula(I)) described herein covalently attaches to the D1 drug moiety via anamine on the piperazine group. In various embodiments, the drug moietyis a derivative of D1. In various embodiments, the D1 derivative retainsat least one biological function or activity as D1 (e.g., SF3b complexbinding, in vitro splicing activity, cytotoxicity) but has an alteredchemical structure.

In some embodiments, the splicing modulator comprises D1:

In some embodiments, the splicing modulator comprises D2:

In some embodiments, the splicing modulator comprises D3:

In some embodiments, the splicing modulator comprises D4:

In some embodiments, the splicing modulator comprises D4′:

In some embodiments, the splicing modulator comprises D5:

In some embodiments, the splicing modulator comprises D6:

In some embodiments, the splicing modulator comprises D7:

In some embodiments, the splicing modulator comprises D8:

In some embodiments, the splicing modulator comprises D9:

In some embodiments, the splicing modulator comprises D10:

In some embodiments, the splicing modulator comprises D11:

In some embodiments, the splicing modulator comprises D12:

In some embodiments, the splicing modulator comprises D13:

In some embodiments, the splicing modulator comprises D14:

In some embodiments, the splicing modulator comprises D15:

In some embodiments, the splicing modulator comprises D16:

In some embodiments, the splicing modulator comprises D17:

In some embodiments, the splicing modulator comprises D18:

In some embodiments, the splicing modulator comprises D19:

In some embodiments, the splicing modulator comprises D20:

In some embodiments, the splicing modulator comprises D21:

In some embodiments, the splicing modulator comprises D22:

In some embodiments, the splicing modulator comprises D23:

In some embodiments, the splicing modulator comprises D24:

In some embodiments, the splicing modulator comprises D25:

In some embodiments, the splicing modulator comprises D26:

In some embodiments, the splicing modulator comprises D27:

In some embodiments, the splicing modulator comprises D28:

In some embodiments, the splicing modulator comprises D29:

In some embodiments, the splicing modulator comprises D30:

In some embodiments, the splicing modulator comprises D31:

In some embodiments, the splicing modulator comprises D32:

In some embodiments, the splicing modulator comprises D33:

In some embodiments, the splicing modulator comprises D34:

In some embodiments, the splicing modulator comprises D35:

An exemplary ADC has Formula (I):

Ab-(L-D)_(p)  (I)

wherein

Ab is an antibody or antigen binding fragment which targets a neoplasticcell;

D is D2;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment targets acell expressing HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B,ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and/or STEAP1.

In some embodiments, the antibody or antigen binding fragment targets aHER2-expressing cell. In some embodiments, the antibody or antigenbinding fragment is an anti-HER2 antibody or antigen binding fragment.In some embodiments, the antibody or antigen binding fragment comprisesthree heavy chain complementarity determining regions (HCDR1, HCDR2, andHCDR3) comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ IDNO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chaincomplementarity determining regions (LCDR1, LCDR2, and LCDR3) comprisingamino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), andSEQ ID NO:6 (LCDR3). In some embodiments, the antibody or antigenbinding fragment comprises a heavy chain variable region comprising anamino acid sequence of SEQ ID NO:19, and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO:20. In some embodiments,the antibody or antigen binding fragment comprises a human IgG1 heavychain constant region. In some embodiments, the antibody or antigenbinding fragment comprises a human Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets a CD138-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CD138 antibody or antigen bindingfragment. In some embodiments, the antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:7(HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11(LCDR2), and SEQ ID NO:12 (LCDR3). In some embodiments, the antibody orantigen binding fragment comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO:21, and a light chainvariable region comprising an amino acid sequence of SEQ ID NO:22. Insome embodiments, the antibody or antigen binding fragment comprises amurine IgG2a heavy chain constant region. In some embodiments, theantibody or antigen binding fragment comprises a murine Ig kappa lightchain constant region. In some embodiments, the antibody or antigenbinding fragment comprises a human IgG2a heavy chain constant region. Insome embodiments, the antibody or antigen binding fragment comprises ahuman Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets an EPHA2-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-EPHA2 antibody or antigen bindingfragment. In some embodiments, the antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:13(HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17(LCDR2), and SEQ ID NO:18 (LCDR3). In some embodiments, the antibody orantigen binding fragment comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO:23, and a light chainvariable region comprising an amino acid sequence of SEQ ID NO:24. Insome embodiments, the antibody or antigen binding fragment comprises ahuman IgG1 heavy chain constant region. In some embodiments, theantibody or antigen binding fragment comprises a human Ig kappa lightchain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets a MSLN-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MSLN antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a FOLH1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-FOLH1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CDH6-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CDH6 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CEACAM5-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CEACAM5 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CFC1B-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CFC1B antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets an ENPP3-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-ENPP3 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a FOLR1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-FOLR1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a HAVCR1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-HAVCR1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a KIT-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-KIT antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a MET-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MET antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a MUC16-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MUC16 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a SLC39A6-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-SLC39A6 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a SLC44A4-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-SLC44A4 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a STEAP1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-STEAP1 antibody or antigen bindingfragment.

In some embodiments, L is selected from any of the linkers disclosedherein, or any combination of linker components disclosed herein. Insome embodiments, L is a linker comprising MC-Val-Cit-pABC,Mal-(PEG)₂-CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB, Mal-Hex,Mal-Et, or Mal-Et-O-Et. In some embodiments, the linker may alsocomprise one or more additional spacer units. In some embodiments, L isan ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, orADL15 linker. In some embodiments, L is an ADL12, ADL14, or ADL15linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12,ADL13, ADL14, ADL21, ADL23, or ADL15 linker may also comprise one ormore additional spacer units.

Another exemplary ADC has Formula (I):

Ab-(L-D)_(p)  (I)

wherein

Ab is an antibody or antigen binding fragment which targets a neoplasticcell;

D is D1;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment targets acell expressing HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B,ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and/or STEAP1.

In some embodiments, the antibody or antigen binding fragment targets aHER2-expressing cell. In some embodiments, the antibody or antigenbinding fragment is an anti-HER2 antibody or antigen binding fragment.In some embodiments, the antibody or antigen binding fragment comprisesthree heavy chain complementarity determining regions (HCDR1, HCDR2, andHCDR3) comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ IDNO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chaincomplementarity determining regions (LCDR1, LCDR2, and LCDR3) comprisingamino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), andSEQ ID NO:6 (LCDR3). In some embodiments, the antibody or antigenbinding fragment comprises a heavy chain variable region comprising anamino acid sequence of SEQ ID NO:19, and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO:20. In some embodiments,the antibody or antigen binding fragment comprises a human IgG1 heavychain constant region. In some embodiments, the antibody or antigenbinding fragment comprises a human Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets a CD138-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CD138 antibody or antigen bindingfragment. In some embodiments, the antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:7(HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11(LCDR2), and SEQ ID NO:12 (LCDR3). In some embodiments, the antibody orantigen binding fragment comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO:21, and a light chainvariable region comprising an amino acid sequence of SEQ ID NO:22. Insome embodiments, the antibody or antigen binding fragment comprises amurine IgG2a heavy chain constant region. In some embodiments, theantibody or antigen binding fragment comprises a murine Ig kappa lightchain constant region. In some embodiments, the antibody or antigenbinding fragment comprises a human IgG2a heavy chain constant region. Insome embodiments, the antibody or antigen binding fragment comprises ahuman Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets an EPHA2-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-EPHA2 antibody or antigen bindingfragment. In some embodiments, the antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:13(HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17(LCDR2), and SEQ ID NO:18 (LCDR3). In some embodiments, the antibody orantigen binding fragment comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO:23, and a light chainvariable region comprising an amino acid sequence of SEQ ID NO:24. Insome embodiments, the antibody or antigen binding fragment comprises ahuman IgG1 heavy chain constant region. In some embodiments, theantibody or antigen binding fragment comprises a human Ig kappa lightchain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets a MSLN-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MSLN antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a FOLH1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-FOLH1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CDH6-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CDH6 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CEACAM5-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CEACAM5 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CFC1B-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CFC1B antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets an ENPP3-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-ENPP3 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a FOLR1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-FOLR1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a HAVCR1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-HAVCR1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a KIT-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-KIT antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a MET-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MET antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a MUC16-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MUC16 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a SLC39A6-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-SLC39A6 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a SLC44A4-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-SLC44A4 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a STEAP1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-STEAP1 antibody or antigen bindingfragment.

Another exemplary ADC has Formula (I):

Ab-(L-D)_(p)  (I)

wherein

Ab is an antibody or antigen binding fragment which targets a neoplasticcell;

D is D4;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment targets acell expressing HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B,ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and/or STEAP1.

In some embodiments, the antibody or antigen binding fragment targets aHER2-expressing cell. In some embodiments, the antibody or antigenbinding fragment is an anti-HER2 antibody or antigen binding fragment.In some embodiments, the antibody or antigen binding fragment comprisesthree heavy chain complementarity determining regions (HCDR1, HCDR2, andHCDR3) comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ IDNO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chaincomplementarity determining regions (LCDR1, LCDR2, and LCDR3) comprisingamino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), andSEQ ID NO:6 (LCDR3). In some embodiments, the antibody or antigenbinding fragment comprises a heavy chain variable region comprising anamino acid sequence of SEQ ID NO:19, and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO:20. In some embodiments,the antibody or antigen binding fragment comprises a human IgG1 heavychain constant region. In some embodiments, the antibody or antigenbinding fragment comprises a human Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets a CD138-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CD138 antibody or antigen bindingfragment. In some embodiments, the antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:7(HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11(LCDR2), and SEQ ID NO:12 (LCDR3). In some embodiments, the antibody orantigen binding fragment comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO:21, and a light chainvariable region comprising an amino acid sequence of SEQ ID NO:22. Insome embodiments, the antibody or antigen binding fragment comprises amurine IgG2a heavy chain constant region. In some embodiments, theantibody or antigen binding fragment comprises a murine Ig kappa lightchain constant region. In some embodiments, the antibody or antigenbinding fragment comprises a human IgG2a heavy chain constant region. Insome embodiments, the antibody or antigen binding fragment comprises ahuman Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets an EPHA2-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-EPHA2 antibody or antigen bindingfragment. In some embodiments, the antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:13(HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17(LCDR2), and SEQ ID NO:18 (LCDR3). In some embodiments, the antibody orantigen binding fragment comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO:23, and a light chainvariable region comprising an amino acid sequence of SEQ ID NO:24. Insome embodiments, the antibody or antigen binding fragment comprises ahuman IgG1 heavy chain constant region. In some embodiments, theantibody or antigen binding fragment comprises a human Ig kappa lightchain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets a MSLN-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MSLN antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a FOLH1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-FOLH1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CDH6-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CDH6 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CEACAM5-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CEACAM5 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CFC1B-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CFC1B antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a ENPP3-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-ENPP3 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a FOLR1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-FOLR1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a HAVCR1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-HAVCR1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a KIT-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-KIT antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a MET-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MET antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a MUC16-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MUC16 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a SLC39A6-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-SLC39A6 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a SLC44A4-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-SLC44A4 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a STEAP1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-STEAP1 antibody or antigen bindingfragment.

Another exemplary ADC has Formula (I):

Ab-(L-D)_(p)  (I)

wherein

Ab is an antibody or antigen binding fragment which targets a neoplasticcell;

D is D12;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment targets acell expressing HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B,ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and/or STEAP1.

In some embodiments, the antibody or antigen binding fragment targets aHER2-expressing cell. In some embodiments, the antibody or antigenbinding fragment is an anti-HER2 antibody or antigen binding fragment.In some embodiments, the antibody or antigen binding fragment comprisesthree heavy chain complementarity determining regions (HCDR1, HCDR2, andHCDR3) comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ IDNO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chaincomplementarity determining regions (LCDR1, LCDR2, and LCDR3) comprisingamino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), andSEQ ID NO:6 (LCDR3). In some embodiments, the antibody or antigenbinding fragment comprises a heavy chain variable region comprising anamino acid sequence of SEQ ID NO:19, and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO:20. In some embodiments,the antibody or antigen binding fragment comprises a human IgG1 heavychain constant region. In some embodiments, the antibody or antigenbinding fragment comprises a human Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets a CD138-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CD138 antibody or antigen bindingfragment. In some embodiments, the antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:7(HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11(LCDR2), and SEQ ID NO:12 (LCDR3). In some embodiments, the antibody orantigen binding fragment comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO:21, and a light chainvariable region comprising an amino acid sequence of SEQ ID NO:22. Insome embodiments, the antibody or antigen binding fragment comprises amurine IgG2a heavy chain constant region. In some embodiments, theantibody or antigen binding fragment comprises a murine Ig kappa lightchain constant region. In some embodiments, the antibody or antigenbinding fragment comprises a human IgG2a heavy chain constant region. Insome embodiments, the antibody or antigen binding fragment comprises ahuman Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets an EPHA2-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-EPHA2 antibody or antigen bindingfragment. In some embodiments, the antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:13(HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17(LCDR2), and SEQ ID NO:18 (LCDR3). In some embodiments, the antibody orantigen binding fragment comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO:23, and a light chainvariable region comprising an amino acid sequence of SEQ ID NO:24. Insome embodiments, the antibody or antigen binding fragment comprises ahuman IgG1 heavy chain constant region. In some embodiments, theantibody or antigen binding fragment comprises a human Ig kappa lightchain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets a MSLN-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MSLN antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a FOLH1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-FOLH1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CDH6-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CDH6 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CEACAM5-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CEACAM5 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CFC1B-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CFC1B antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a ENPP3-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-ENPP3 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a FOLR1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-FOLR1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a HAVCR1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-HAVCR1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a KIT-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-KIT antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a MET-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MET antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a MUC16-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MUC16 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a SLC39A6-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-SLC39A6 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a SLC44A4-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-SLC44A4 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a STEAP1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-STEAP1 antibody or antigen bindingfragment.

Another exemplary ADC has Formula (I):

Ab-(L-D)_(p)  (I)

wherein

Ab is an antibody or antigen binding fragment which targets a neoplasticcell;

D is D15;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment targets acell expressing HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B,ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and/or STEAP1.

In some embodiments, the antibody or antigen binding fragment targets aHER2-expressing cell. In some embodiments, the antibody or antigenbinding fragment is an anti-HER2 antibody or antigen binding fragment.In some embodiments, the antibody or antigen binding fragment comprisesthree heavy chain complementarity determining regions (HCDR1, HCDR2, andHCDR3) comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ IDNO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chaincomplementarity determining regions (LCDR1, LCDR2, and LCDR3) comprisingamino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), andSEQ ID NO:6 (LCDR3). In some embodiments, the antibody or antigenbinding fragment comprises a heavy chain variable region comprising anamino acid sequence of SEQ ID NO:19, and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO:20. In some embodiments,the antibody or antigen binding fragment comprises a human IgG1 heavychain constant region. In some embodiments, the antibody or antigenbinding fragment comprises a human Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets a CD138-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CD138 antibody or antigen bindingfragment. In some embodiments, the antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:7(HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11(LCDR2), and SEQ ID NO:12 (LCDR3). In some embodiments, the antibody orantigen binding fragment comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO:21, and a light chainvariable region comprising an amino acid sequence of SEQ ID NO:22. Insome embodiments, the antibody or antigen binding fragment comprises amurine IgG2a heavy chain constant region. In some embodiments, theantibody or antigen binding fragment comprises a murine Ig kappa lightchain constant region. In some embodiments, the antibody or antigenbinding fragment comprises a human IgG2a heavy chain constant region. Insome embodiments, the antibody or antigen binding fragment comprises ahuman Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets an EPHA2-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-EPHA2 antibody or antigen bindingfragment. In some embodiments, the antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:13(HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17(LCDR2), and SEQ ID NO:18 (LCDR3). In some embodiments, the antibody orantigen binding fragment comprises a heavy chain variable regioncomprising an amino acid sequence of SEQ ID NO:23, and a light chainvariable region comprising an amino acid sequence of SEQ ID NO:24. Insome embodiments, the antibody or antigen binding fragment comprises ahuman IgG1 heavy chain constant region. In some embodiments, theantibody or antigen binding fragment comprises a human Ig kappa lightchain constant region.

In some other embodiments, the antibody or antigen binding fragmenttargets a MSLN-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MSLN antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a FOLH1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-FOLH1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CDH6-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CDH6 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CEACAM5-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CEACAM5 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a CFC1B-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-CFC1B antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a ENPP3-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-ENPP3 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a FOLR1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-FOLR1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a HAVCR1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-HAVCR1 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a KIT-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-KIT antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a MET-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MET antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a MUC16-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-MUC16 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a SLC39A6-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-SLC39A6 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a SLC44A4-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-SLC44A4 antibody or antigen bindingfragment.

In some other embodiments, the antibody or antigen binding fragmenttargets a STEAP1-expressing cell. In some embodiments, the antibody orantigen binding fragment is an anti-STEAP1 antibody or antigen bindingfragment.

In some embodiments, L is selected from any of the linkers disclosedherein, or any combination of linker components disclosed herein. Insome embodiments, L is a cleavable linker. In some embodiments, L is anon-cleavable linker. In some embodiments, L is a linker comprisingMC-Val-Cit-pABC, MC-Val-Ala-pABC, MC-Val-Ala-pAB, MC-Glu-Val-Cit-pABC,Ala-Ala-Asn-pABC, or β-glucuronide. In some embodiments, L is a linkercomprising Mal-Hex, Mal-Et, or Mal-Et-O-Et. In some embodiments, L is alinker comprising MC-Val-Cit-pABC, Mal-(PEG)₂-CO, MC-Val-Ala-pAB,MC-Val-Ala-pABC, MC-Val-Cit-pAB, Mal-Hex, Mal-Et, or Mal-Et-O-Et. Insome embodiments, the linker may also comprise one or more additionalspacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7,ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker. In some embodiments,L is an ADL12, ADL14, or ADL15 linker. In some embodiments, the ADL1,ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15linker may also comprise one or more additional spacer units.

In some embodiments, p is from 1 to 10. In some embodiments, p is from 2to 8. In some embodiments, p is from 4 to 8. In some embodiments, p is4. In some embodiments, p is 8.

Another exemplary ADC has Formula (I):

Ab-(L-D)_(p)  (I)

wherein Ab is an anti-HER2 antibody or antigen binding fragmentcomprising three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:1(HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5(LCDR2), and SEQ ID NO:6 (LCDR3);

D is D2, D1, D4, D12, or D15;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment comprisesa heavy chain variable region comprising an amino acid sequence of SEQID NO:19, and a light chain variable region comprising an amino acidsequence of SEQ ID NO:20.

In some embodiments, L is selected from any of the linkers disclosedherein, or any combination of linker components disclosed herein. Insome embodiments, L is a linker comprising MC-Val-Cit-pABC,Mal-(PEG)₂-CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB, Mal-Hex,Mal-Et, or Mal-Et-O-Et. In some embodiments, the linker may alsocomprise one or more additional spacer units. In some embodiments, L isan ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, orADL15 linker. In some embodiments, L is an ADL12, ADL14, or ADL15linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12,ADL13, ADL14, ADL21, ADL23, or ADL15 linker may also comprise one ormore additional spacer units.

Another exemplary ADC has Formula (I):

Ab-(L-D)_(p)  (I)

wherein Ab is an anti-CD138 antibody or antigen binding fragmentcomprising three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:7(HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11(LCDR2), and SEQ ID NO:12 (LCDR3);

D is D2, D1, D4, D12, or D15;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment comprisesa heavy chain variable region comprising an amino acid sequence of SEQID NO:21, and a light chain variable region comprising an amino acidsequence of SEQ ID NO:22.

In some embodiments, L is selected from any of the linkers disclosedherein, or any combination of linker components disclosed herein. Insome embodiments, L is a linker comprising MC-Val-Cit-pABC,Mal-(PEG)₂-CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB, Mal-Hex,Mal-Et, or Mal-Et-O-Et. In some embodiments, the linker may alsocomprise one or more additional spacer units. In some embodiments, L isan ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, orADL15 linker. In some embodiments, L is an ADL12, ADL14, or ADL15linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12,ADL13, ADL14, ADL21, ADL23, or ADL15 linker may also comprise one ormore additional spacer units.

Another exemplary ADC has Formula (I):

Ab-(L-D)_(p)  (I)

wherein Ab is an anti-EPHA2 antibody or antigen binding fragmentcomprising three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:13(HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17(LCDR2), and SEQ ID NO:18 (LCDR3);

D is D2, D1, D4, D12, or D15;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment comprisesa heavy chain variable region comprising an amino acid sequence of SEQID NO:23, and a light chain variable region comprising an amino acidsequence of SEQ ID NO:24.

In some embodiments, L is selected from any of the linkers disclosedherein, or any combination of linker components disclosed herein. Insome embodiments, L is a linker comprising MC-Val-Cit-pABC,Mal-(PEG)₂-CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB, Mal-Hex,Mal-Et, or Mal-Et-O-Et. In some embodiments, the linker may alsocomprise one or more additional spacer units. In some embodiments, L isan ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, orADL15 linker. In some embodiments, L is an ADL12, ADL14, or ADL15linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12,ADL13, ADL14, ADL21, ADL23, or ADL15 linker may also comprise one ormore additional spacer units.

In various embodiments, ADCs comprising a D2 or D1 drug moiety caninclude a cleavable or non-cleavable linker.

In various embodiments, the linker is a cleavable linker. In variousembodiments, the cleavable linker comprises MC-Val-Cit-pABC. In variousembodiments, the cleavable linker comprises MC-Val-Ala-pABC. In variousembodiments, the cleavable linker comprises MC-Val-Ala-pAB. In variousembodiments, the cleavable linker comprises MC-Glu-Val-Cit-pABC. Invarious embodiments, the cleavable linker comprises MC-Ala-Ala-Asn-pABC.

In various other embodiments, the linker is a non-cleavable linker. Invarious embodiments, the non-cleavable linker comprises MC, alone or incombination with at least one additional spacer unit. In variousembodiments, the non-cleavable linker comprises Mal-Hex, alone or incombination with at least one additional spacer unit. In variousembodiments, the non-cleavable linker comprises Mal-Et, alone or incombination with at least one additional spacer unit. In variousembodiments, the non-cleavable linker comprises Mal-Et-O-Et, alone or incombination with at least one additional spacer unit.

In various embodiments, in ADCs comprising a D2 or D1 drug moiety, p isfrom 1 to 10. In various embodiments, p is from 2 to 8. In variousembodiments, p is from 4 to 8. In various embodiments, p is 4. Invarious embodiments, p is 8.

In various embodiments, an ADC comprising a D2 or D1 drug moiety asdisclosed herein demonstrates improved drug:antibody ratio, loweraggregation levels, increased stability, increased on-target killing ofcancer cells, decreased off-target killing of non-cancer cells, and/orincreased cytotoxicity and/or potency relative to an ADC comprising analternate drug moiety (e.g., an alternate splicing modulator drugmoiety). In some embodiments, an ADC comprising a D2 or D1 drug moietyas disclosed herein provides good or superior properties in one or moreof the categories listed above, or across a spectrum of functionalproperties for a therapeutic ADC. In some embodiments, an ADC comprisinga D2 or D1 drug moiety exhibits surprisingly effective potency andincreased inhibition of cell growth and/or proliferation in cells thatexpress the antigen targeted by the ADC, as compared to an ADCcomprising an alternate drug moiety (e.g., an alternate splicingmodulator drug moiety). In some embodiments, potency can be measured interms of the concentration of compound to cause 50% reduction in cellproliferation (GI₅₀). In various embodiments, ADCs comprising a D2 or D1drug moiety exhibit surprisingly increased in vivo stability (e.g.,plasma stability), as compared to an ADC with an alternate drug moiety(e.g., an alternate splicing modulator drug moiety, e.g., thailanstatinA). See, e.g., the ADC described in Puthenveetil et al. (BioconjugateChem. (2016) 27:1880-8), which shows complete bioconversion of payloadby 72 hours (i.e., acetate is completely hydrolyzed).

In certain embodiments, an intermediate, which is the precursor of thelinker moiety, is reacted with the drug moiety (e.g., the splicingmodulator) under appropriate conditions. In certain embodiments,reactive groups are used on the drug and/or the intermediate or linker.The product of the reaction between the drug and the intermediate, orthe derivatized drug (drug plus linker), is subsequently reacted withthe antibody or antigen binding fragment under appropriate conditions.Alternatively, the intermediate or linker may first be reacted with theantibody or antigen binding fragment, or a derivatized antibody orantigen binding fragment, and then reacted with the drug or derivatizeddrug.

A number of different reactions are available for covalent attachment ofthe drug moiety and/or linker moiety to the antibody or antigen bindingfragment. This is often accomplished by reaction of one or more aminoacid residues of the antibody or antigen binding fragment, including theamine groups of lysine, the free carboxylic acid groups of glutamic acidand aspartic acid, the sulfhydryl groups of cysteine, and the variousmoieties of the aromatic amino acids. For instance, non-specificcovalent attachment may be undertaken using a carbodiimide reaction tolink a carboxy (or amino) group on a drug moiety to an amino (orcarboxy) group on an antibody or antigen binding fragment. Additionally,bifunctional agents such as dialdehydes or imidoesters may also be usedto link the amino group on a drug moiety to an amino group on anantibody or antigen binding fragment. Also available for attachment ofdrugs (e.g., a splicing modulator) to binding agents is the Schiff basereaction. This method involves the periodate oxidation of a drug thatcontains glycol or hydroxy groups, thus forming an aldehyde which isthen reacted with the binding agent. Attachment occurs via formation ofa Schiff base with amino groups of the binding agent. Isothiocyanatesmay also be used as coupling agents for covalently attaching drugs tobinding agents. Other techniques are known to the skilled artisan andwithin the scope of the present disclosure. Examples of drug moietiesthat can be generated and linked to an antibody or antigen bindingfragment using various chemistries known to in the art include splicingmodulators, e.g., the splicing modulators described and exemplifiedherein.

Linker-Drug/Drug Compounds

Further disclosed herein are exemplary linker-drug (L-D) compounds, aswell as compositions comprising multiple copies of such compounds. Invarious embodiments, the linker-drug compounds disclosed herein can bedefined by the generic formula: L-D, wherein L=a linker moiety, and D=adrug moiety (e.g., a splicing modulator drug moiety). In certainembodiments, the disclosed L-D compounds are suitable for use in theADCs described herein, e.g., in ADCs of Formula (I).

In various embodiments, the linker-drug (L-D) compounds disclosed hereincomprise a linker-drug structure according to Formula (III). In variousembodiments, the present disclosure provides a linker-drug (L-D)compound of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is chosen from absent, hydrogen, C₁-C₆ alkyl groups, C₁-C₆alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acidgroups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzylgroups, C₃-C₈ heterocyclyl groups, —O—C(═O)—(C₁-C₈ alkyl) groups, and—CD₃;

R² is absent or a linker; R³ is chosen from hydrogen, C₁-C₆ alkylgroups, C₁-C₆ alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆alkylcarboxylic acid groups, C₁-C₆ alkylhydroxy groups, C₃-C₆ cycloalkylgroups, benzyl groups, C₃-C₈ heterocyclyl groups, and —O—C(═O)—(C₁-C₆alkyl) groups; and

R⁴, R⁵, and R⁸ are each independently chosen from hydrogen, hydroxylgroups, —O—(C₁-C₆ alkyl) groups, —O—C(═O)—(C₁-C₈ alkyl) groups, andC₁-C₈ alkyl groups;

R⁶ and R⁷ are each independently chosen from hydrogen, —O—R¹⁷,—O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₈ alkyl groups, —NR¹⁵R¹⁶, and alinker;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷;

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups; and

Z″ is chosen from

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, R¹⁶, and R¹⁷ are eachindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups,—NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆alkylalkoxy groups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein at least one of R⁶ and R⁷ is hydrogen; and

wherein if R² is a linker, then neither R⁶ or R⁷ is a linker, and if R⁶or R⁷ is a linker, then R² is absent.

In some embodiments, R¹ is chosen from absent, hydrogen, C₁-C₄ alkylgroups, C₁-C₄ alkylcarboxylic acid groups, and C₃-C₈ cycloalkyl groups.In some embodiments, R¹ is absent. In some embodiments, R¹ is hydrogen.In some embodiments, R¹ is a C₁-C₄ alkyl group. In some embodiments, R¹is methyl. In some embodiments, R¹ is ethyl. In some embodiments, R¹ isa C₁-C₄ alkylcarboxylic acid group. In some embodiments, R¹ is—CH₂CH₂CH₂CO₂H. In some embodiments, R¹ is a C₃-C₈ cycloalkyl group. Insome embodiments, R¹ is cycloheptyl.

In some embodiments, R³ is chosen from hydrogen, C₁-C₄ alkyl groups,C₁-C₄ alkylalkoxy groups, C₁-C₄ alkylcarboxylic acid groups, and C₁-C₄alkylhydroxy groups. In some embodiments, R³ is chosen from hydrogen andC₁-C₄ alkylcarboxylic acid groups. In some embodiments, R³ is hydrogen.In some embodiments, R³ is a C₁-C₄ alkylcarboxylic acid group. In someembodiments, R³ is —CH₂CH₂CO₂H.

In some embodiments, R⁴ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, —O—C(═O)—(C₁-C₄ alkyl) groups, and C₁-C₄ alkylgroups. In some embodiments, R⁴ is hydrogen. In some embodiments, R⁴ ishydroxyl. In some embodiments, R⁴ is a —O—(C₁-C₄ alkyl) group. In someembodiments, R⁴ is —OCH₃. In some embodiments, R⁴ is —OCH₂CH₃. In someembodiments, R⁴ is a —O—C(═O)—(C₁-C₄ alkyl) group. In some embodiments,R⁴ is —O—C(═O)—CH₃. In some embodiments, R⁴ is —O—C(═O)—CH₂CH₃. In someembodiments, R⁴ is a C₁-C₄ alkyl group. In some embodiments, R⁴ ismethyl. In some embodiments, R⁴ is ethyl.

In some embodiments, R⁵ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, and C₁-C₄ alkyl groups. In some embodiments, R⁵is hydrogen. In some embodiments, R⁵ is hydroxyl. In some embodiments,R⁵ is a —O—(C₁-C₄ alkyl) group. In some embodiments, R⁵ is a C₁-C₄ alkylgroup.

In some embodiments, R² is absent and R⁶ is a linker. In someembodiments, R² is absent and R⁷ is a linker. In some embodiments, R² isa linker. In some embodiments, R⁶ is hydrogen. In some embodiments, R⁷is hydrogen.

In some embodiments, R⁶ is hydrogen and R⁷ is —O—R¹⁷. In someembodiments, R⁶ is hydrogen and R⁷ is —OR¹⁷, wherein R¹⁷ is chosen fromhydrogen and C¹-C⁴ alkyl groups. In some embodiments, R⁶ is hydrogen andR⁷ is —O—R¹⁷, wherein R¹⁷ is hydrogen. In some embodiments, R⁶ is —O—R¹⁷and R⁷ is hydrogen. In some embodiments, R⁶ is —O—R¹⁷ and R⁷ ishydrogen, wherein R¹⁷ is chosen from hydrogen and C¹-C⁴ alkyl groups. Insome embodiments, R⁶ is —O—R¹⁷ and R⁷ is hydrogen, wherein R¹⁷ ishydrogen. In some embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶. Insome embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶, wherein R¹⁵ is Hand R¹⁶ is chosen from hydrogen, R¹⁷, —C(═O)—R¹⁷, and —C(═O)—O—R¹⁷. Insome embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶, wherein R¹⁵ is Hand R¹⁶ is chosen from hydrogen, R¹⁷, —C(═O)—R¹⁷, and —C(═O)—O—R¹⁷, andwherein R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In some embodiments,R⁶ is —O—R¹⁷. In some embodiments, R⁶ is —O—C(═O)—R¹⁷. In someembodiments, R⁶ is C₁-C₆ alkyl. In some embodiments, R⁶ is C₁-C₄ alkyl.In some embodiments, R⁶ is C₁ alkyl. In some embodiments, R⁶ is—NR¹⁵R¹⁶.

In some embodiments, R⁷ is —O—R¹⁷. In some embodiments, R⁷ is—O—C(═O)—R¹⁷. In some embodiments, R⁷ is C₁-C₆ alkyl. In someembodiments, R⁷ is C₁-C₄ alkyl. In some embodiments, R⁷ is C₁ alkyl. Insome embodiments, R⁷ is —NR¹⁵R¹⁶.

In some embodiments, R⁸ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, and (C₁-C₄ alkyl). In some embodiments, R⁸ ishydrogen. In some embodiments, R⁸ is a hydroxyl group. In someembodiments, R⁸ is an —O—(C₁-C₄ alkyl) group. In some embodiments, R⁸ isan —O—(C₁ alkyl) group.

In some embodiments, R¹⁵ is hydrogen. In some embodiments, R¹⁵ is R¹⁷.In some embodiments, R¹⁵ is —C(═O)—R¹⁷. In some embodiments, R¹⁵ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁶ is hydrogen. In some embodiments, R¹⁶ is R¹⁷.In some embodiments, R¹⁶ is —C(═O)—R¹⁷. In some embodiments, R¹⁶ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁷ is chosen from hydrogen, C₁-C₄ alkyl groups,C₃-C₆ cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In someembodiments, R¹⁷ is hydrogen. In some embodiments, R¹⁷ is a C₁-C₄ alkylgroup. In some embodiments, R¹⁷ is a C₁ alkyl group. In someembodiments, R¹⁷ is a C₃-C₆ cycloalkyl group. In some embodiments, R¹⁷is a C₃ cycloalkyl group. In some embodiments, R¹⁷ is a C₄ cycloalkylgroup. In some embodiments, R¹⁷ is a C₅ cycloalkyl group. In someembodiments, R¹⁷ is a C₆ cycloalkyl group. In some embodiments, R¹⁷ is aC₃-C₈ heterocyclyl group. In some embodiments, R¹⁷ is a C₃ heterocyclylgroup. In some embodiments, R¹⁷ is a C₄ heterocyclyl group. In someembodiments, R¹⁷ is a C₅ heterocyclyl group. In some embodiments, R¹⁷ isa C₆ heterocyclyl group. In some embodiments, R¹⁷ is a C₇ heterocyclylgroup. In some embodiments, R¹⁷ is a C₈ heterocyclyl group.

In some embodiments, Z′ is

In some embodiments, Z′ is

In some embodiments, Z′ is

In some embodiments, Z′ is

In some embodiments, Z′ is

In some embodiments, R² is a linker. In some embodiments, the linkercomprises at least one cleavable peptide moiety. In some embodiments,the at least one cleavable peptide moiety is cleavable by an enzyme. Insome embodiments, the linker or cleavable peptide moiety comprises atleast one amino acid unit. In some embodiments, the at least one aminoacid unit is chosen from arginine, histidine, lysine, aspartic acid,glutamic acid, serine, threonine, asparagine, glutamine, cysteine,selenocysteine, glycine, proline, alanine, valine, isoleucine,methionine, phenylalanine, tyrosine, tryptophan, and citrulline. In someembodiments, the at least one amino acid unit is chosen from alanine,citrulline, and valine. In some embodiments, the linker comprisescitrulline and valine. In some embodiments, the linker comprises alanineand valine.

In some embodiments, the linker comprises a moiety chosen from asulfonamide, a β-glucuronide, a disulfide, and a carbonyl. In someembodiments, the linker comprises a sulfonamide. In some embodiments,the linker comprises a β-glucuronide. In some embodiments, the linkercomprises a disulfide. In some embodiments, the linker comprises acarbonyl.

In some embodiments, the linker comprises a spacer unit. In someembodiments, the spacer unit is chosen from alkyl groups andpolyethylene glycol (PEG) moieties. In some embodiments, the alkyl groupis a C₁-C₁₂ alkyl group. In some embodiments, the alkyl group is a C₁-C₆alkyl group. In some embodiments, the alkyl group is methylene. In someembodiments, the alkyl group is ethylene. In some embodiments, the alkylgroup is n-propylene. In some embodiments, the alkyl group isn-butylene. In some embodiments, the alkyl group is n-pentylene. In someembodiments, the alkyl group is n-hexylene. In some embodiments, the PEGmoiety comprises -(PEG)_(m)-, wherein m is an integer from 1 to 10. Insome embodiments, m is 1. In some embodiments, m is 2. In someembodiments, m is 3. In some embodiments, m is 4. In some embodiments, mis 5. In some embodiments, m is 6.

In some embodiments, the linker comprises a maleimide (Mal) moiety(“Mal-spacer unit”). In some embodiments, the linker comprises aself-immolative spacer unit. In some embodiments, the self-immolativespacer unit is chosen from p-aminobenzyloxycarbonyl (pABC) andp-aminobenzyl (pAB).

In some embodiments, the linker comprises a Mal-spacer unit, an alkylgroup, at least one amino acid unit, and a self-immolative spacer. Insome embodiments, at least one amino acid unit is chosen from alanine,citrulline, and valine. In some embodiments, the at least one amino acidunit comprises alanine and valine. In some embodiments, the at least oneamino acid unit comprises citrulline and valine. In some embodiments,the self-immolative spacer is chosen from pAB and pABC. In someembodiments, the self-immolative spacer comprises pAB. In someembodiments, the self-immolative spacer comprises pABC. In someembodiments, the alkyl group comprises a C₁-C₆ alkyl group.

In some embodiments, the linker comprises a Mal-spacer unit, PEG moiety,at least one amino acid unit, and a self-immolative spacer. In someembodiments, at least one amino acid unit is chosen from alanine,citrulline, and valine. In some embodiments, the at least one amino acidunit comprises alanine and valine. In some embodiments, the at least oneamino acid unit comprises citrulline and valine. In some embodiments,the self-immolative spacer is chosen from pAB and pABC. In someembodiments, the self-immolative spacer comprises pAB. In someembodiments, the self-immolative spacer comprises pABC. In someembodiments, the PEG moiety comprises -(PEG)_(m)-, wherein m is aninteger from 1 to 6.

In various other embodiments, the linker-drug (L-D) compounds disclosedherein comprise a linker-drug structure according to Formula (V). Invarious embodiments, the present disclosure provides a linker-drug (L-D)compound of Formula (V):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ is chosen from absent, hydrogen, C₁-C₆ alkyl groups, C₁-C₆alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acidgroups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzylgroups, C₃-C₈ heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl) groups, and—CD₃;

R² is absent or a linker; R³ is chosen from hydrogen, C₁-C₆ alkylgroups, C₁-C₆ alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆alkylcarboxylic acid groups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkylgroups, benzyl groups, C₃-C₈ heterocyclyl groups, and —O—C(═O)—(C₁-C₆alkyl) groups; and

R⁴, R⁵, and R⁸ are each independently chosen from hydrogen, hydroxylgroups, —O—(C₁-C₆ alkyl) groups, —O—C(═O)—(C₁-C₆ alkyl) groups, andC₁-C₆ alkyl groups;

R⁶ and R⁷ are each independently chosen from hydrogen, —O—R¹⁷,—O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₆ alkyl groups, —NR¹⁵R¹⁶, and alinker;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷; and

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, R¹⁶, and R¹⁷ are eachindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups,—NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆alkylalkoxy groups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein at least one of R⁶ and R⁷ is hydrogen; and

wherein if R² is a linker, then neither R⁶ or R⁷ is a linker, and if R⁶or R⁷ is a linker, then R² is absent.

In some embodiments, R¹ is chosen from absent, hydrogen, C₁-C₄ alkylgroups, C₁-C₄ alkylcarboxylic acid groups, and C₃-C₈ cycloalkyl groups.In some embodiments, R¹ is absent. In some embodiments, R¹ is hydrogen.In some embodiments, R¹ is a C₁-C₄ alkyl group. In some embodiments, R¹is methyl. In some embodiments, R¹ is ethyl. In some embodiments, R¹ isa C₁-C₄ alkylcarboxylic acid group. In some embodiments, R¹ is—CH₂CH₂CH₂CO₂H. In some embodiments, R¹ is a C₃-C₈ cycloalkyl group. Insome embodiments, R¹ is cycloheptyl.

In some embodiments, R³ is chosen from hydrogen, C₁-C₄ alkyl groups,C₁-C₄ alkylalkoxy groups, C₁-C₄ alkylcarboxylic acid groups, and C₁-C₄alkylhydroxy groups. In some embodiments, R³ is chosen from hydrogen andC₁-C₄ alkylcarboxylic acid groups. In some embodiments, R³ is hydrogen.In some embodiments, R³ is a C₁-C₄ alkylcarboxylic acid group. In someembodiments, R³ is —CH₂CH₂CO₂H.

In some embodiments, R⁴ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, —O—C(═O)—(C₁-C₄ alkyl) groups, and C₁-C₄ alkylgroups. In some embodiments, R⁴ is hydrogen. In some embodiments, R⁴ ishydroxyl. In some embodiments, R⁴ is a —O—(C₁-C₄ alkyl) group. In someembodiments, R⁴ is —OCH₃. In some embodiments, R⁴ is —OCH₂CH₃. In someembodiments, R⁴ is a —O—C(═O)—(C₁-C₄ alkyl) group. In some embodiments,R⁴ is —O—C(═O)—CH₃. In some embodiments, R⁴ is —O—C(═O)—CH₂CH₃. In someembodiments, R⁴ is a C₁-C₄ alkyl group. In some embodiments, R⁴ ismethyl. In some embodiments, R⁴ is ethyl.

In some embodiments, R⁵ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, and C₁-C₄ alkyl groups. In some embodiments, R⁵is hydrogen. In some embodiments, R⁵ is hydroxyl. In some embodiments,R⁵ is a —O—(C₁-C₄ alkyl) group. In some embodiments, R⁵ is a C₁-C₄ alkylgroup.

In some embodiments, R² is absent and R⁶ is a linker. In someembodiments, R² is absent and R⁷ is a linker. In some embodiments, R² isa linker. In some embodiments, R⁶ is hydrogen. In some embodiments, R⁷is hydrogen.

In some embodiments, R⁶ is hydrogen and R⁷ is —O—R¹⁷. In someembodiments, R⁶ is hydrogen and R⁷ is —OR¹⁷, wherein R¹⁷ is chosen fromhydrogen and C¹-C⁴ alkyl groups. In some embodiments, R⁶ is hydrogen andR⁷ is —O—R¹⁷, wherein R¹⁷ is hydrogen. In some embodiments, R⁶ is —O—R¹⁷and R⁷ is hydrogen. In some embodiments, R⁶ is —O—R¹⁷ and R⁷ ishydrogen, wherein R¹⁷ is chosen from hydrogen and C¹-C⁴ alkyl groups. Insome embodiments, R⁶ is —O—R¹⁷ and R⁷ is hydrogen, wherein R¹⁷ ishydrogen. In some embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶. Insome embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶, wherein R¹⁵ is Hand R¹⁶ is chosen from hydrogen, R¹⁷, —C(═O)—R¹⁷, and —C(═O)—O—R¹⁷. Insome embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶, wherein R¹⁵ is Hand R¹⁶ is chosen from hydrogen, R¹⁷, —C(═O)—R¹⁷, and —C(═O)—O—R¹⁷, andwherein R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In some embodiments,R⁶ is —O—R¹⁷. In some embodiments, R⁶ is —O—C(═O)—R¹⁷. In someembodiments, R⁶ is C₁-C₆ alkyl. In some embodiments, R⁶ is C₁-C₄ alkyl.In some embodiments, R⁶ is C₁ alkyl. In some embodiments, R⁶ is—NR¹⁵R¹⁶.

In some embodiments, R⁷ is —O—R¹⁷. In some embodiments, R⁷ is—O—C(═O)—R¹⁷. In some embodiments, R⁷ is C₁-C₆ alkyl. In someembodiments, R⁷ is C₁-C₄ alkyl. In some embodiments, R⁷ is C₁ alkyl. Insome embodiments, R⁷ is —NR¹⁵R¹⁶.

In some embodiments, R⁸ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, and (C₁-C₄ alkyl). In some embodiments, R⁸ ishydrogen. In some embodiments, R⁸ is a hydroxyl group. In someembodiments, R⁸ is an —O—(C₁-C₄ alkyl) group. In some embodiments, R⁸ isan —O—(C₁ alkyl) group.

In some embodiments, R¹⁵ is hydrogen. In some embodiments, R¹⁵ is R¹⁷.In some embodiments, R¹⁵ is —C(═O)—R¹⁷. In some embodiments, R¹⁵ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁶ is hydrogen. In some embodiments, R¹⁶ is R¹⁷.In some embodiments, R¹⁶ is —C(═O)—R¹⁷. In some embodiments, R¹⁶ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁷ is chosen from hydrogen, C₁-C₄ alkyl groups,C₃-C₆ cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In someembodiments, R¹⁷ is hydrogen. In some embodiments, R¹⁷ is a C₁-C₄ alkylgroup. In some embodiments, R¹⁷ is a C₁ alkyl group. In someembodiments, R¹⁷ is a C₃-C₆ cycloalkyl group. In some embodiments, R¹⁷is a C₃ cycloalkyl group. In some embodiments, R¹⁷ is a C₄ cycloalkylgroup. In some embodiments, R¹⁷ is a C₅ cycloalkyl group. In someembodiments, R¹⁷ is a C₆ cycloalkyl group. In some embodiments, R¹⁷ is aC₃-C₈ heterocyclyl group. In some embodiments, R¹⁷ is a C₃ heterocyclylgroup. In some embodiments, R¹⁷ is a C₄ heterocyclyl group. In someembodiments, R¹⁷ is a C₅ heterocyclyl group. In some embodiments, R¹⁷ isa C₆ heterocyclyl group. In some embodiments, R¹⁷ is a C₇ heterocyclylgroup. In some embodiments, R¹⁷ is a C₈ heterocyclyl group.

In some embodiments, R² is a linker. In some embodiments, the linkercomprises at least one cleavable peptide moiety. In some embodiments,the at least one cleavable peptide moiety is cleavable by an enzyme. Insome embodiments, the linker or cleavable peptide moiety comprises atleast one amino acid unit. In some embodiments, the at least one aminoacid unit is chosen from arginine, histidine, lysine, aspartic acid,glutamic acid, serine, threonine, asparagine, glutamine, cysteine,selenocysteine, glycine, proline, alanine, valine, isoleucine,methionine, phenylalanine, tyrosine, tryptophan, and citrulline. In someembodiments, the at least one amino acid unit is chosen from alanine,citrulline, and valine. In some embodiments, the linker comprisescitrulline and valine. In some embodiments, the linker comprises alanineand valine.

In some embodiments, the linker comprises a moiety chosen from asulfonamide, a β-glucuronide, a disulfide, and a carbonyl. In someembodiments, the linker comprises a sulfonamide. In some embodiments,the linker comprises a β-glucuronide. In some embodiments, the linkercomprises a disulfide. In some embodiments, the linker comprises acarbonyl.

In some embodiments, the linker comprises a spacer unit. In someembodiments, the spacer unit is chosen from alkyl groups andpolyethylene glycol (PEG) moieties. In some embodiments, the alkyl groupis a C₁-C₁₂ alkyl group. In some embodiments, the alkyl group is a C₁-C₆alkyl group. In some embodiments, the alkyl group is methylene. In someembodiments, the alkyl group is ethylene. In some embodiments, the alkylgroup is n-propylene. In some embodiments, the alkyl group isn-butylene. In some embodiments, the alkyl group is n-pentylene. In someembodiments, the alkyl group is n-hexylene. In some embodiments, the PEGmoiety comprises -(PEG)_(n)-, wherein m is an integer from 1 to 10. Insome embodiments, m is 1. In some embodiments, m is 2. In someembodiments, m is 3. In some embodiments, m is 4. In some embodiments, mis 5. In some embodiments, m is 6.

In some embodiments, the linker comprises a maleimide (Mal) moiety(“Mal-spacer unit”). In some embodiments, the linker comprises aself-immolative spacer unit. In some embodiments, the self-immolativespacer unit is chosen from p-aminobenzyloxycarbonyl (pABC) andp-aminobenzyl (pAB).

In some embodiments, the linker comprises a Mal-spacer unit, an alkylgroup, at least one amino acid unit, and a self-immolative spacer. Insome embodiments, at least one amino acid unit is chosen from alanine,citrulline, and valine. In some embodiments, the at least one amino acidunit comprises alanine and valine. In some embodiments, the at least oneamino acid unit comprises citrulline and valine. In some embodiments,the self-immolative spacer is chosen from pAB and pABC. In someembodiments, the self-immolative spacer comprises pAB. In someembodiments, the self-immolative spacer comprises pABC. In someembodiments, the alkyl group comprises a C₁-C₆ alkyl group.

In some embodiments, the linker comprises a Mal-spacer unit, PEG moiety,at least one amino acid unit, and a self-immolative spacer. In someembodiments, at least one amino acid unit is chosen from alanine,citrulline, and valine. In some embodiments, the at least one amino acidunit comprises alanine and valine. In some embodiments, the at least oneamino acid unit comprises citrulline and valine. In some embodiments,the self-immolative spacer is chosen from pAB and pABC. In someembodiments, the self-immolative spacer comprises pAB. In someembodiments, the self-immolative spacer comprises pABC. In someembodiments, the PEG moiety comprises -(PEG)_(m)-, wherein m is aninteger from 1 to 6.

In various other embodiments, the linker-drug (L-D) compounds disclosedherein comprise a linker-drug structure according to Formula (VII). Invarious embodiments, the present disclosure provides a linker-drug (L-D)compound of Formula (VII):

or a pharmaceutically acceptable salt thereof, wherein:

R¹ and R⁹ are each independently chosen from absent, hydrogen, C₁-C₆alkyl groups, C₁-C₆ alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆alkylcarboxylic acid groups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkylgroups, benzyl groups, C₃-C₈ heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl)groups, and —CD₃;

R² is absent or a linker; R³ is chosen from hydrogen, C₁-C₆ alkylgroups, C₁-C₆ alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆alkylcarboxylic acid groups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkylgroups, benzyl groups, C₃-C₈ heterocyclyl groups, and —O—C(═O)—(C₁-C₆alkyl) groups;

R⁴, R⁵, and R⁸ are each independently chosen from hydrogen, hydroxylgroups, —O—(C₁-C₆ alkyl) groups, —O—C(═O)—(C₁-C₆ alkyl) groups, andC₁-C₆ alkyl groups;

R⁶ and R⁷ are each independently chosen from hydrogen, —O—R¹⁷,—O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₆ alkyl groups, —NR¹⁵R¹⁶, and alinker;

R¹⁰ is chosen from hydrogen, C₁-C₆ alkyl groups, —C(═O)—(C₁-C₆ alkyl)groups, and —CD₃;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷;

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups; and

a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹⁵, R¹⁶, and R¹⁷ areeach independently substituted with 0 to 3 groups independently chosenfrom halogens, hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl)groups, —NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups,C₁-C₆ alkylalkoxy groups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein at least one of R⁶ and R⁷ is hydrogen;

wherein if R² is a linker, then neither R⁶ or R⁷ is a linker, and if R⁶or R⁷ is a linker, then R² is absent; and

wherein R¹ and R⁹ cannot both be absent.

In some embodiments, R¹ is chosen from absent, hydrogen, C₁-C₄ alkylgroups, C₁-C₄ alkylcarboxylic acid groups, and C₃-C₈ cycloalkyl groups.In some embodiments, R¹ is absent. In some embodiments, R¹ is hydrogen.In some embodiments, R¹ is a C₁-C₄ alkyl group. In some embodiments, R¹is methyl. In some embodiments, R¹ is ethyl. In some embodiments, R¹ isa C₁-C₄ alkylcarboxylic acid group. In some embodiments, R¹ is—CH₂CH₂CH₂CO₂H. In some embodiments, R¹ is a C₃-C₈ cycloalkyl group. Insome embodiments, R¹ is cycloheptyl.

In some embodiments, R³ is chosen from hydrogen, C₁-C₄ alkyl groups,C₁-C₄ alkylalkoxy groups, C₁-C₄ alkylcarboxylic acid groups, and C₁-C₄alkylhydroxy groups. In some embodiments, R³ is chosen from hydrogen andC₁-C₄ alkylcarboxylic acid groups. In some embodiments, R³ is hydrogen.In some embodiments, R³ is a C₁-C₄ alkylcarboxylic acid group. In someembodiments, R³ is —CH₂CH₂CO₂H.

In some embodiments, R⁴ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, —O—C(═O)—(C₁-C₄ alkyl) groups, and C₁-C₄ alkylgroups. In some embodiments, R⁴ is hydrogen. In some embodiments, R⁴ ishydroxyl. In some embodiments, R⁴ is a —O—(C₁-C₄ alkyl) group. In someembodiments, R⁴ is —OCH₃. In some embodiments, R⁴ is —OCH₂CH₃. In someembodiments, R⁴ is a —O—C(═O)—(C₁-C₄ alkyl) group. In some embodiments,R⁴ is —O—C(═O)—CH₃. In some embodiments, R⁴ is —O—C(═O)—CH₂CH₃. In someembodiments, R⁴ is a C₁-C₄ alkyl group. In some embodiments, R⁴ ismethyl. In some embodiments, R⁴ is ethyl.

In some embodiments, R⁵ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, and C₁-C₄ alkyl groups. In some embodiments, R⁵is hydrogen. In some embodiments, R⁵ is hydroxyl. In some embodiments,R⁵ is a —O—(C₁-C₄ alkyl) group. In some embodiments, R⁵ is a C₁-C₄ alkylgroup.

In some embodiments, R⁹ is chosen from absent, hydrogen, C₁-C₄ alkylgroups, —(C═O)—(C₁-C₄ alkyl) groups, and —CD₃. In some embodiments, R⁹is absent. In some embodiments, R⁹ is hydrogen. In some embodiments, R⁹is a C₁-C₄ alkyl group. In some embodiments, the C₁-C₄ alkyl group ismethyl. In some embodiments, the C₁-C₄ alkyl group is ethyl. In someembodiments, R⁹ is a —(C═O)—(C₁-C₄ alkyl) group. In some embodiments,the —(C═O)—(C₁-C₄ alkyl) group is —(C═O)-methyl. In some embodiments, R⁹is —CD₃.

In some embodiments, R¹⁰ is chosen from hydrogen, C₁-C₄ alkyl groups,—(C═O)—(C₁-C₄ alkyl) groups, and —CD₃. In some embodiments, R¹⁰ ishydrogen. In some embodiments, R¹⁰ is a C₁-C₄ alkyl group. In someembodiments, the C₁-C₄ alkyl group is methyl. In some embodiments, theC₁-C₄ alkyl group is ethyl. In some embodiments, R¹⁰ is a —(C═O)—(C₁-C₄alkyl) group. In some embodiments, the —(C═O)—(C₁-C₄ alkyl) group is—(C═O)-methyl. In some embodiments, R¹⁰ is —CD₃.

In some embodiments, R² is absent and R⁶ is a linker. In someembodiments, R² is absent and R⁷ is a linker. In some embodiments, R² isa linker. In some embodiments, R⁶ is hydrogen. In some embodiments, R⁷is hydrogen. In some embodiments, R⁶ is hydrogen and R⁷ is —O—R¹⁷. Insome embodiments, R⁶ is hydrogen and R⁷ is —OR¹⁷, wherein R¹⁷ is chosenfrom hydrogen and C¹-C⁴ alkyl groups. In some embodiments, R⁶ ishydrogen and R⁷ is —O—R¹⁷, wherein R¹⁷ is hydrogen. In some embodiments,R⁶ is —O—R¹⁷ and R⁷ is hydrogen. In some embodiments, R⁶ is —O—R¹⁷ andR⁷ is hydrogen, wherein R¹⁷ is chosen from hydrogen and C¹-C⁴ alkylgroups. In some embodiments, R⁶ is —O—R¹⁷ and R⁷ is hydrogen, whereinR¹⁷ is hydrogen. In some embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶.In some embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶, wherein R¹⁵ is Hand R¹⁶ is chosen from hydrogen, R¹⁷, —C(═O)—R¹⁷, and —C(═O)—O—R¹⁷. Insome embodiments, R⁶ is hydrogen and R⁷ is —NR¹⁵R¹⁶, wherein R¹⁵ is Hand R¹⁶ is chosen from hydrogen, R¹⁷, —C(═O)—R¹⁷, and —C(═O)—O—R¹⁷, andwherein R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In some embodiments,R⁶ is —O—R¹⁷. In some embodiments, R⁶ is —O—C(═O)—R¹⁷. In someembodiments, R⁶ is C₁-C₆ alkyl. In some embodiments, R⁶ is C₁-C₄ alkyl.In some embodiments, R⁶ is C₁ alkyl. In some embodiments, R⁶ is—NR¹⁵R¹⁶.

In some embodiments, R⁷ is —O—R¹⁷. In some embodiments, R⁷ is—O—C(═O)—R¹⁷. In some embodiments, R⁷ is C₁-C₆ alkyl. In someembodiments, R⁷ is C₁-C₄ alkyl. In some embodiments, R⁷ is C₁ alkyl. Insome embodiments, R⁷ is —NR¹⁵R¹⁶.

In some embodiments, R⁸ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, and (C₁-C₄ alkyl). In some embodiments, R⁸ ishydrogen. In some embodiments, R⁸ is a hydroxyl group. In someembodiments, R⁸ is an —O—(C₁-C₄ alkyl) group. In some embodiments, R⁸ isan —O—(C₁ alkyl) group.

In some embodiments, R¹⁵ is hydrogen. In some embodiments, R¹⁵ is R¹⁷.In some embodiments, R¹⁵ is —C(═O)—R¹⁷. In some embodiments, R¹⁵ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁶ is hydrogen. In some embodiments, R¹⁶ is R¹⁷.In some embodiments, R¹⁶ is —C(═O)—R¹⁷. In some embodiments, R¹⁶ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁷ is chosen from hydrogen, C₁-C₄ alkyl groups,C₃-C₆ cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In someembodiments, R¹⁷ is hydrogen. In some embodiments, R¹⁷ is a C₁-C₄ alkylgroup. In some embodiments, R¹⁷ is a C₁ alkyl group. In someembodiments, R¹⁷ is a C₃-C₆ cycloalkyl group. In some embodiments, R¹⁷is a C₃ cycloalkyl group. In some embodiments, R¹⁷ is a C₄ cycloalkylgroup. In some embodiments, R¹⁷ is a C₅ cycloalkyl group. In someembodiments, R¹⁷ is a C₆ cycloalkyl group. In some embodiments, R¹⁷ is aC₃-C₈ heterocyclyl group. In some embodiments, R¹⁷ is a C₃ heterocyclylgroup. In some embodiments, R¹⁷ is a C₄ heterocyclyl group. In someembodiments, R¹⁷ is a C₅ heterocyclyl group. In some embodiments, R¹⁷ isa C₆ heterocyclyl group. In some embodiments, R¹⁷ is a C₇ heterocyclylgroup. In some embodiments, R¹⁷ is a C₈ heterocyclyl group.

In some embodiments, a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In someembodiments, a is 1, 2, 3, 4, 5, or 6. In some embodiments, a is 1, 2,3, 4, or 5. In some embodiments, a is 1, 2, 3, or 4. In someembodiments, a is 1, 2, or 3. In some embodiments, a is 1 or 2. In someembodiments, a is 1. In some embodiments, a is 2. In some embodiments, ais 3. In some embodiments, a is 4. In some embodiments, a is 5. In someembodiments, a is 6. In some embodiments, a is 7. In some embodiments, ais 8. In some embodiments, a is 9. In some embodiments, a is 10.

In some embodiments, R² is a linker. In some embodiments, the linkercomprises at least one cleavable peptide moiety. In some embodiments,the at least one cleavable peptide moiety is cleavable by an enzyme. Insome embodiments, the linker or cleavable peptide moiety comprises atleast one amino acid unit. In some embodiments, the at least one aminoacid unit is chosen from arginine, histidine, lysine, aspartic acid,glutamic acid, serine, threonine, asparagine, glutamine, cysteine,selenocysteine, glycine, proline, alanine, valine, isoleucine,methionine, phenylalanine, tyrosine, tryptophan, and citrulline. In someembodiments, the at least one amino acid unit is chosen from alanine,citrulline, and valine. In some embodiments, the linker comprisescitrulline and valine. In some embodiments, the linker comprises alanineand valine.

In some embodiments, the linker comprises a moiety chosen from asulfonamide, a β-glucuronide, a disulfide, and a carbonyl. In someembodiments, the linker comprises a sulfonamide. In some embodiments,the linker comprises a β-glucuronide. In some embodiments, the linkercomprises a disulfide. In some embodiments, the linker comprises acarbonyl.

In some embodiments, the linker comprises a spacer unit. In someembodiments, the spacer unit is chosen from alkyl groups andpolyethylene glycol (PEG) moieties. In some embodiments, the alkyl groupis a C₁-C₁₂ alkyl group. In some embodiments, the alkyl group is a C₁-C₆alkyl group. In some embodiments, the alkyl group is methylene. In someembodiments, the alkyl group is ethylene. In some embodiments, the alkylgroup is n-propylene. In some embodiments, the alkyl group isn-butylene. In some embodiments, the alkyl group is n-pentylene. In someembodiments, the alkyl group is n-hexylene. In some embodiments, the PEGmoiety comprises -(PEG)_(m)-, wherein m is an integer from 1 to 10. Insome embodiments, m is 1. In some embodiments, m is 2. In someembodiments, m is 3. In some embodiments, m is 4. In some embodiments, mis 5. In some embodiments, m is 6.

In some embodiments, the linker comprises a maleimide (Mal) moiety(“Mal-spacer unit”). In some embodiments, the linker comprises aself-immolative spacer unit. In some embodiments, the self-immolativespacer unit is chosen from p-aminobenzyloxycarbonyl (pABC) andp-aminobenzyl (pAB).

In some embodiments, the linker comprises a Mal-spacer unit, an alkylgroup, at least one amino acid unit, and a self-immolative spacer. Insome embodiments, at least one amino acid unit is chosen from alanine,citrulline, and valine. In some embodiments, the at least one amino acidunit comprises alanine and valine. In some embodiments, the at least oneamino acid unit comprises citrulline and valine. In some embodiments,the self-immolative spacer is chosen from pAB and pABC. In someembodiments, the self-immolative spacer comprises pAB. In someembodiments, the self-immolative spacer comprises pABC. In someembodiments, the alkyl group comprises a C₁-C₆ alkyl group.

In some embodiments, the linker comprises a Mal-spacer unit, PEG moiety,at least one amino acid unit, and a self-immolative spacer. In someembodiments, at least one amino acid unit is chosen from alanine,citrulline, and valine. In some embodiments, the at least one amino acidunit comprises alanine and valine. In some embodiments, the at least oneamino acid unit comprises citrulline and valine. In some embodiments,the self-immolative spacer is chosen from pAB and pABC. In someembodiments, the self-immolative spacer comprises pAB. In someembodiments, the self-immolative spacer comprises pABC. In someembodiments, the PEG moiety comprises -(PEG)_(m)-, wherein m is aninteger from 1 to 6.

In various other embodiments, the linker-drug (L-D) compounds disclosedherein comprise a linker-drug structure according to Formula (IX). Invarious embodiments, the present disclosure provides a linker-drug (L-D)compound of Formula (IX):

or a pharmaceutically acceptable salt thereof, wherein: R¹ is chosenfrom absent, hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxy groups,C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups, C₁-C₆alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl) groups, and —CD₃;

R² is a linker; R³ is chosen from hydrogen, C₁-C₆ alkyl groups, C₁-C₆alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acidgroups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzylgroups, C₃-C₈ heterocyclyl groups, and —O—C(═O)—(C₁-C₆ alkyl) groups;

R⁴ is chosen from hydrogen, hydroxyl groups, —O—(C₁-C₆ alkyl) groups,—O—C(═O)—(C₁-C₆ alkyl) groups, and C₁-C₆ alkyl groups;

R¹⁰ is chosen from 3 to 10 membered carbocycles and 3 to 10 memberedheterocycles, each of which is substituted with 0 to 3 R^(a), whereineach R^(a) is independently chosen from halogens, C₁-C₆ alkyl groups,—O—(C₁-C₆)alkyl groups, C₁-C₆ alkylalkoxy groups, C₁-C₆ alkylhydroxygroups, —S(═O)_(w)-(4 to 7 membered heterocycles), 4 to 7 memberedcarbocycles, and 4 to 7 membered heterocycles;

R¹⁵ and R¹⁶ are each independently chosen from hydrogen, R¹⁷,—C(═O)—R¹⁷, and —C(═O)—O—R¹⁷; and

R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkylgroups, benzyl groups, and C₃-C₈ heterocyclyl groups;

wherein R¹, R², R³, R⁴, R¹⁰, R¹⁵, R¹⁶, and R¹⁷ are each independentlysubstituted with 0 to 3 groups independently chosen from halogens,hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups, —NR¹⁵R¹⁶,C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆ alkylalkoxygroups, benzyl groups, and C₃-C₈ heterocyclyl groups; and wherein eachR^(a) is independently substituted with 0 to 3 groups independentlychosen from halogens, hydroxyl groups, —NR¹⁵R¹⁶, C₁-C₆ alkyl groups,—(C═O)—(C₁-C₆ alkyl) groups, —(C═O)—(C₁-C₆ alkyl)-(C₃-C₁₀ heterocyclylgroups), and C₁-C₆ alkylcarboxylic acid groups, each of which issubstituted with 0, 1, or 2 groups independently chosen from halogens,hydroxyl groups, —NR¹⁵R¹⁶, and C₁-C₃ alkyl groups; and

w is 0, 1, or 2.

In some embodiments, R¹ is chosen from absent, hydrogen, C₁-C₄ alkylgroups, C₁-C₄ alkylcarboxylic acid groups, and C₃-C₈ cycloalkyl groups.In some embodiments, R¹ is absent. In some embodiments, R¹ is hydrogen.In some embodiments, R¹ is a C₁-C₄ alkyl group. In some embodiments, R¹is methyl. In some embodiments, R¹ is ethyl. In some embodiments, R¹ isa C₁-C₄ alkylcarboxylic acid group. In some embodiments, R¹ is—CH₂CH₂CH₂CO₂H. In some embodiments, R¹ is a C₃-C₈ cycloalkyl group. Insome embodiments, R¹ is cycloheptyl.

In some embodiments, R³ is chosen from hydrogen, C₁-C₄ alkyl groups,C₁-C₄ alkylalkoxy groups, C₁-C₄ alkylcarboxylic acid groups, and C₁-C₄alkylhydroxy groups. In some embodiments, R³ is chosen from hydrogen andC₁-C₄ alkylcarboxylic acid groups. In some embodiments, R³ is hydrogen.In some embodiments, R³ is a C₁-C₄ alkylcarboxylic acid group. In someembodiments, R³ is —CH₂CH₂CO₂H.

In some embodiments, R⁴ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, —O—C(═O)—(C₁-C₄ alkyl) groups, and C₁-C₄ alkylgroups. In some embodiments, R⁴ is hydrogen. In some embodiments, R⁴ ishydroxyl. In some embodiments, R⁴ is a —O—(C₁-C₄ alkyl) group. In someembodiments, R⁴ is —OCH₃. In some embodiments, R⁴ is —OCH₂CH₃. In someembodiments, R⁴ is a —O—C(═O)—(C₁-C₄ alkyl) group. In some embodiments,R⁴ is —O—C(═O)—CH₃. In some embodiments, R⁴ is —O—C(═O)—CH₂CH₃. In someembodiments, R⁴ is a C₁-C₄ alkyl group. In some embodiments, R⁴ ismethyl. In some embodiments, R⁴ is ethyl.

In some embodiments, R¹⁰ is chosen from 6 to 9 membered carbocycles and6 to 9 membered heterocycles, each of which is substituted with 0 to 2R^(a), wherein each R^(a) is independently substituted with 0 to 3groups independently chosen from halogens, hydroxyl groups, C₁-C₆ alkylgroups, —(C═O)—(C₁-C₆ alkyl) groups, —(C═O)—(C₁-C₆ alkyl)-(3 to 10membered heterocycle) groups, and C₁-C₆ alkylcarboxylic acid groups.

In some embodiments, the carbocycle is a phenyl substituted with 0 to 2R^(a), wherein each R^(a) is independently substituted with 0 to 3groups independently chosen from halogens, hydroxyl groups, C₁-C₆ alkylgroups, —(C═O)—(C₁-C₆ alkyl) groups, —(C═O)—(C₁-C₆ alkyl)-(3 to 10membered heterocycle) groups, and C₁-C₆ alkylcarboxylic acid groups. Insome embodiments, the phenyl is substituted with 2 R^(a), wherein eachR^(a) is independently substituted with 0 to 3 groups independentlychosen from halogens, hydroxyl groups, C₁-C₆ alkyl groups, —(C═O)—(C₁-C₆alkyl) groups, —(C═O)—(C₁-C₆ alkyl)-(3 to 10 membered heterocycle)groups, and C₁-C₆ alkylcarboxylic acid groups. In some embodiments, thephenyl is

In some embodiments, the heterocycle is a 9 membered heterocyclesubstituted with 0 to 2 R^(a), wherein each R^(a) is independentlysubstituted with 0 to 3 groups independently chosen from halogens,hydroxyl groups, C₁-C₆ alkyl groups, —(C═O)—(C₁-C₆ alkyl) groups,—(C═O)—(C₁-C₆ alkyl)-(3 to 10 membered heterocycle) groups, and C₁-C₆alkylcarboxylic acid groups. In some embodiments, the 9 memberedheterocycle is

In some embodiments, R^(a) is chosen from halogens, 3 to 10 memberedcarbocycles, and 3 to 10 membered heterocycles, wherein each R^(a) isindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups, —(C═O)—(C₁-C₆ alkyl)groups, —(C═O)—(C₁-C₆ alkyl)-(3 to 10 membered heterocycle) groups, andC₁-C₆ alkylcarboxylic acid groups. In some embodiments, R^(a) is chosenfrom halogens,

In some embodiments, R¹⁵ is hydrogen. In some embodiments, R¹⁵ is R¹⁷.In some embodiments, R¹⁵ is —C(═O)—R¹⁷. In some embodiments, R¹⁵ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁶ is hydrogen. In some embodiments, R¹⁶ is R¹⁷.In some embodiments, R¹⁶ is —C(═O)—R¹⁷. In some embodiments, R¹⁶ is—C(═O)—O—R¹⁷.

In some embodiments, R¹⁷ is chosen from hydrogen, C₁-C₄ alkyl groups,C₃-C₆ cycloalkyl groups, and C₃-C₈ heterocyclyl groups. In someembodiments, R¹⁷ is hydrogen. In some embodiments, R¹⁷ is a C₁-C₄ alkylgroup. In some embodiments, R¹⁷ is a C₁ alkyl group. In someembodiments, R¹⁷ is a C₃-C₆ cycloalkyl group. In some embodiments, R¹⁷is a C₃ cycloalkyl group. In some embodiments, R¹⁷ is a C₄ cycloalkylgroup. In some embodiments, R¹⁷ is a C₅ cycloalkyl group. In someembodiments, R¹⁷ is a C₆ cycloalkyl group. In some embodiments, R¹⁷ is aC₃-C₈ heterocyclyl group. In some embodiments, R¹⁷ is a C₃ heterocyclylgroup. In some embodiments, R¹⁷ is a C₄ heterocyclyl group. In someembodiments, R¹⁷ is a C₅ heterocyclyl group. In some embodiments, R¹⁷ isa C₆ heterocyclyl group. In some embodiments, R¹⁷ is a C₇ heterocyclylgroup. In some embodiments, R¹⁷ is a C₈ heterocyclyl group.

In some embodiments, R² is a linker. In some embodiments, the linkercomprises at least one cleavable peptide moiety. In some embodiments,the at least one cleavable peptide moiety is cleavable by an enzyme. Insome embodiments, the linker or cleavable peptide moiety comprises atleast one amino acid unit. In some embodiments, the at least one aminoacid unit is chosen from arginine, histidine, lysine, aspartic acid,glutamic acid, serine, threonine, asparagine, glutamine, cysteine,selenocysteine, glycine, proline, alanine, valine, isoleucine,methionine, phenylalanine, tyrosine, tryptophan, and citrulline. In someembodiments, the at least one amino acid unit is chosen from alanine,citrulline, and valine. In some embodiments, the linker comprisescitrulline and valine. In some embodiments, the linker comprises alanineand valine.

In some embodiments, the linker comprises a moiety chosen from asulfonamide, a β-glucuronide, a disulfide, and a carbonyl. In someembodiments, the linker comprises a sulfonamide. In some embodiments,the linker comprises a β-glucuronide. In some embodiments, the linkercomprises a disulfide. In some embodiments, the linker comprises acarbonyl.

In some embodiments, the linker comprises a spacer unit. In someembodiments, the spacer unit is chosen from alkyl groups andpolyethylene glycol (PEG) moieties. In some embodiments, the alkyl groupis a C₁-C₁₂ alkyl group. In some embodiments, the alkyl group is a C₁-C₆alkyl group. In some embodiments, the alkyl group is methylene. In someembodiments, the alkyl group is ethylene. In some embodiments, the alkylgroup is n-propylene. In some embodiments, the alkyl group isn-butylene. In some embodiments, the alkyl group is n-pentylene. In someembodiments, the alkyl group is n-hexylene. In some embodiments, the PEGmoiety comprises -(PEG)_(m)-, wherein m is an integer from 1 to 10. Insome embodiments, m is 1. In some embodiments, m is 2. In someembodiments, m is 3. In some embodiments, m is 4. In some embodiments, mis 5. In some embodiments, m is 6.

In some embodiments, the linker comprises a maleimide (Mal) moiety(“Mal-spacer unit”). In some embodiments, the linker comprises aself-immolative spacer unit.

In some embodiments, the self-immolative spacer unit is chosen fromp-aminobenzyloxycarbonyl (pABC) and p-aminobenzyl (pAB).

In some embodiments, the linker comprises a Mal-spacer unit, an alkylgroup, at least one amino acid unit, and a self-immolative spacer. Insome embodiments, at least one amino acid unit is chosen from alanine,citrulline, and valine. In some embodiments, the at least one amino acidunit comprises alanine and valine. In some embodiments, the at least oneamino acid unit comprises citrulline and valine. In some embodiments,the self-immolative spacer is chosen from pAB and pABC. In someembodiments, the self-immolative spacer comprises pAB. In someembodiments, the self-immolative spacer comprises pABC. In someembodiments, the alkyl group comprises a C₁-C₆ alkyl group.

In some embodiments, the linker comprises a Mal-spacer unit, PEG moiety,at least one amino acid unit, and a self-immolative spacer. In someembodiments, at least one amino acid unit is chosen from alanine,citrulline, and valine. In some embodiments, the at least one amino acidunit comprises alanine and valine. In some embodiments, the at least oneamino acid unit comprises citrulline and valine. In some embodiments,the self-immolative spacer is chosen from pAB and pABC. In someembodiments, the self-immolative spacer comprises pAB. In someembodiments, the self-immolative spacer comprises pABC. In someembodiments, the PEG moiety comprises -(PEG)_(m)-, wherein m is aninteger from 1 to 6.

In some embodiments, the drug moiety is a splicing modulator chosen fromD1, D2, D3, D4, D4′, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15,D16, D17, D18, D19, D20, D21, D22, D23, D24, D25, D26, D27, D28, D29,D30, D31, D32, D33, D34, and D35.

In various embodiments, the drug moiety is a splicing modulator selectedfrom D2 and D1.

In various embodiments, the drug moiety is D2. In various embodiments,the structure of the D2 drug moiety used in the disclosed linker-drug(L-D) compounds is shown below:

In various embodiments, the linker in the linker-drug (L-D) compoundsdescribed herein covalently attaches to the D2 drug moiety via an amineon the piperazine group. In various embodiments, the drug moiety is aderivative of D2. In various embodiments, the D2 derivative retains atleast one biological function or activity as D2 (e.g., SF3b complexbinding, in vitro splicing activity, cytotoxicity) but has an alteredchemical structure.

In various embodiments, the drug moiety is D1. In various embodiments,the structure of the D1 drug moiety used in the disclosed linker-drug(L-D) compounds is shown below:

In various embodiments, the linker in the linker-drug (L-D) compoundsdescribed herein covalently attaches to the D1 drug moiety via an amineon the piperazine group. In various embodiments, the drug moiety is aderivative of D1. In various embodiments, the D1 derivative retains atleast one biological function or activity as D1 (e.g., SF3b complexbinding, in vitro splicing activity, cytotoxicity) but has an alteredchemical structure

Also disclosed herein are exemplary splicing modulator drug compounds,for use on their own or as drug moieties in the ADCs disclosed herein.The present disclosure, in various embodiments, further providescompositions comprising multiple copies of such compounds, e.g.,pharmaceutical compositions comprising a splicing modulator drugcompound and a pharmaceutically acceptable carrier.

In various embodiments, the present disclosure provides a compoundchosen from a compound of formula

and pharmaceutically acceptable salts thereof.

In various embodiments, the present disclosure further provides apharmaceutical composition comprising the compound, or apharmaceutically acceptable salt thereof, and a pharmaceuticallyacceptable carrier.

In various other embodiments, the present disclosure provides a compoundchosen from a compound of formula

and pharmaceutically acceptable salts thereof. In various embodiments,the present disclosure further provides a pharmaceutical compositioncomprising the compound, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

In various other embodiments, the present disclosure provides a compoundchosen from a compound of formula

and pharmaceutically acceptable salts thereof. In various embodiments,the present disclosure further provides a pharmaceutical compositioncomprising the compound, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

In various other embodiments, the present disclosure provides a compoundchosen from a compound of formula

and pharmaceutically acceptable salts thereof. In various embodiments,the present disclosure further provides a pharmaceutical compositioncomprising the compound, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

In various other embodiments, the present disclosure provides a compoundchosen from a compound of formula

and pharmaceutically acceptable salts thereof. In various embodiments,the present disclosure further provides a pharmaceutical compositioncomprising the compound, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

In various other embodiments, the present disclosure provides a compoundchosen from a compound of formula

and pharmaceutically acceptable salts thereof. In various embodiments,the present disclosure further provides a pharmaceutical compositioncomprising the compound, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

In various other embodiments, the present disclosure provides a compoundchosen from a compound of formula

and pharmaceutically acceptable salts thereof. In various embodiments,the present disclosure further provides a pharmaceutical compositioncomprising the compound, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

In various other embodiments, the present disclosure provides a compoundchosen from a compound of formula

and pharmaceutically acceptable salts thereof. In various embodiments,the present disclosure further provides a pharmaceutical compositioncomprising the compound, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

In various other embodiments, the present disclosure provides a compoundchosen from a compound of formula

and pharmaceutically acceptable salts thereof. In various embodiments,the present disclosure further provides a pharmaceutical compositioncomprising the compound, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

In various other embodiments, the present disclosure provides a compoundchosen from a compound of formula

and pharmaceutically acceptable salts thereof. In various embodiments,the present disclosure further provides a pharmaceutical compositioncomprising the compound, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

In various other embodiments, the present disclosure provides a compoundchosen from a compound of formula

and pharmaceutically acceptable salts thereof. In various embodiments,the present disclosure further provides a pharmaceutical compositioncomprising the compound, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

In various other embodiments, the present disclosure provides a compoundchosen from a compound of formula

and pharmaceutically acceptable salts thereof. In various embodiments,the present disclosure further provides a pharmaceutical compositioncomprising the compound, or a pharmaceutically acceptable salt thereof,and a pharmaceutically acceptable carrier.

Bioconjugation

In some embodiments, a linker-drug compound may be conjugated to anantibody or antigen binding fragment according to the exemplary schemedepicted in FIG. 26. Briefly, in some embodiments, an antibody orantigen binding fragment may be treated with a reagent, e.g., a reducingagent, e.g., tris(2-carboxyethyl)phosphine, to activate the antibody orantigen binding fragment by reducing one or more disulfide bonds. Insome embodiments, the activated antibody or antigen binding fragment isthen treated with a linker-drug compound at a predeterminedstoichiometry. Subsequently, in some embodiments, the mixture is thensubjected to a purification technique, e.g., size exclusion resin orultrafiltration to afford the desired antibody-drug conjugate.

In some embodiments, provided herein is an antibody-drug conjugatehaving the structure

wherein Ab is an antibody or antigen binding fragment covalently boundto a maleimide group of the linker-drug compound (ADL1-D1) through thesulfur atom of a thiol group on the antibody or antigen bindingfragment.

In some embodiments, provided herein is an antibody-drug conjugatehaving the structure

wherein Ab is an antibody or antigen binding fragment covalently boundto a maleimide group of the linker-drug compound (ADL1-D4) through thesulfur atom of a thiol group on the antibody or antigen bindingfragment.

In some embodiments, provided herein is an antibody-drug conjugatehaving the structure

wherein Ab is an antibody or antigen binding fragment covalently boundto a maleimide group of the linker-drug compound (ADL1-D12) through thesulfur atom of a thiol group on the antibody or antigen bindingfragment.

In some embodiments, provided herein is an antibody-drug conjugatehaving the structure

wherein Ab is an antibody or antigen binding fragment covalently boundto a maleimide group of the linker-drug compound (ADL5-D2) through thesulfur atom of a thiol group on the antibody or antigen bindingfragment.

In some embodiments, provided herein is an antibody-drug conjugatehaving the structure

wherein Ab is an antibody or antigen binding fragment covalently boundto a maleimide group of the linker-drug compound (ADL5-D15) through thesulfur atom of a thiol group on the antibody or antigen bindingfragment.

In some embodiments, provided herein is an antibody-drug conjugatehaving the structure

wherein Ab is an antibody or antigen binding fragment covalently boundto a maleimide group of the linker-drug compound (ADL13-D4) through thesulfur atom of a thiol group on the antibody or antigen bindingfragment.

In some embodiments, the Ab is an anti-HER2 antibody or an antigenbinding fragment thereof. In some embodiments, the Ab binds to HER2 andtargets HER2-expressing neoplastic cells (i.e., the ADC targetsHER2-expressing neoplastic cells). In some embodiments, the Ab is aninternalizing anti-HER2 antibody or internalizing antigen bindingfragment thereof.

In some embodiments, the anti-HER2 antibody or antigen binding fragmentcomprises three heavy chain complementarity determining regions (HCDR1,HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:1(HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three lightchain complementarity determining regions (LCDR1, LCDR2, and LCDR3)comprising amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5(LCDR2), and SEQ ID NO:6 (LCDR3). In some embodiments, the anti-HER2antibody or antigen binding fragment is an internalizing antibody orinternalizing antigen binding fragment. In some embodiments, theanti-HER2 antibody or antigen binding fragment comprises human frameworksequences. In some embodiments, the anti-HER2 antibody or antigenbinding fragment comprises a heavy chain variable region comprising anamino acid sequence of SEQ ID NO:19, and a light chain variable regioncomprising an amino acid sequence of SEQ ID NO:20. In some embodiments,the anti-HER2 antibody or antigen binding fragment comprises a humanIgG1 heavy chain constant region. In some embodiments, the anti-HER2antibody or antigen binding fragment comprises a human Ig kappa lightchain constant region.

Drug Loading

Drug loading is represented by p, and is also referred to herein as thedrug-to-antibody ratio (DAR). Drug loading may range from 1 to 10 drugmoieties per antibody or antigen binding fragment. In some embodiments,p is an integer from 1 to 10. In some embodiments, p is an integer from1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to2. In some embodiments, p is an integer from 2 to 10, 2 to 9, 2 to 8, 2to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p is aninteger from 1 to 8. In some embodiments, p is an integer from 2 to 4.In other embodiments, p is an integer from 4 to 8. In other embodiments,p is 1, 2, 3, 4, 5, 6, 7, or 8, preferably 4 or 8.

Drug loading may be limited by the number of attachment sites on theantibody or antigen binding fragment. In some embodiments, the linkermoiety (L) of the ADC attaches to the antibody or antigen bindingfragment through a chemically active group on one or more amino acidresidues on the antibody or antigen binding fragment. For example, thelinker may be attached to the antibody or antigen binding fragment via afree amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N-or C-terminus, to the epsilon amino group of one or more lysineresidues, to the free carboxylic acid group of one or more glutamic acidor aspartic acid residues, or to the sulfhydryl group of one or morecysteine residues). The site to which the linker is attached can be anatural residue in the amino acid sequence of the antibody or antigenbinding fragment, or it can be introduced into the antibody or antigenbinding fragment, e.g., by DNA recombinant technology (e.g., byintroducing a cysteine residue into the amino acid sequence) or byprotein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).

In some embodiments, the number of drug moieties that can be conjugatedto an antibody or antigen binding fragment is limited by the number offree cysteine residues. For example, where the attachment is a cysteinethiol group, an antibody may have only one or a few cysteine thiolgroups, or may have only one or a few sufficiently reactive thiol groupsthrough which a linker may be attached. Generally, antibodies do notcontain many free and reactive cysteine thiol groups that may be linkedto a drug moiety. Indeed, most cysteine thiol residues in antibodies areinvolved in either interchain or intrachain disulfide bonds. Conjugationto cysteines can, in some embodiments, therefore require at leastpartial reduction of the antibody. Over-attachment of linker-toxin to anantibody may destabilize the antibody by reducing the cysteine residuesavailable to form disulfide bonds. Therefore, an optimal drug:antibodyratio should increase potency of the ADC (by increasing the number ofattached drug moieties per antibody) without destabilizing the antibodyor antigen binding fragment. In some embodiments, an optimal ratio maybe 2, 4, 6, or 8.

In some embodiments, an antibody or antigen binding fragment is exposedto reducing conditions prior to conjugation in order to generate one ormore free cysteine residues. An antibody, in some embodiments, may bereduced with a reducing agent such as dithiothreitol (DTT) ortris(2-carboxyethyl)phosphine (TCEP), under partial or total reducingconditions, to generate reactive cysteine thiol groups. Unpairedcysteines may be generated through partial reduction with limited molarequivalents of TCEP, which can reduce the interchain disulfide bondswhich link the light chain and heavy chain (one pair per H-L pairing)and the two heavy chains in the hinge region (two pairs per H-H pairingin the case of human IgG1) while leaving the intrachain disulfide bondsintact (Stefano et al. (2013) Methods Mol Biol. 1045:145-71). Inembodiments, disulfide bonds within the antibodies are reducedelectrochemically, e.g., by employing a working electrode that appliesan alternating reducing and oxidizing voltage. This approach can allowfor on-line coupling of disulfide bond reduction to an analytical device(e.g., an electrochemical detection device, an NMR spectrometer, or amass spectrometer) or a chemical separation device (e.g., a liquidchromatograph (e.g., an HPLC) or an electrophoresis device (see, e.g.,U.S. Publ. No. 20140069822)). In certain embodiments, an antibody issubjected to denaturing conditions to reveal reactive nucleophilicgroups on amino acid residues, such as cysteine.

The drug loading of an ADC may be controlled in different ways, e.g.,by: (i) limiting the molar excess of drug-linker intermediate or linkerreagent relative to antibody; (ii) limiting the conjugation reactiontime or temperature; (iii) partial or limiting reductive conditions forcysteine thiol modification; and/or (iv) engineering by recombinanttechniques the amino acid sequence of the antibody such that the numberand position of cysteine residues is modified for control of the numberand/or position of linker-drug attachments.

In some embodiments, free cysteine residues are introduced into theamino acid sequence of the antibody or antigen binding fragment. Forexample, cysteine engineered antibodies can be prepared wherein one ormore amino acids of a parent antibody are replaced with a cysteine aminoacid. Any form of antibody may be so engineered, i.e. mutated. Forexample, a parent Fab antibody fragment may be engineered to form acysteine engineered Fab referred to as a “ThioFab.” Similarly, a parentmonoclonal antibody may be engineered to form a “ThioMab.” A single sitemutation yields a single engineered cysteine residue in a ThioFab,whereas a single site mutation yields two engineered cysteine residuesin a ThioMab, due to the dimeric nature of the IgG antibody. DNAencoding an amino acid sequence variant of the parent polypeptide can beprepared by a variety of methods known in the art (see, e.g., themethods described in Intl. Pub. No. WO 2006/034488). These methodsinclude, but are not limited to, preparation by site-directed (oroligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassettemutagenesis of an earlier prepared DNA encoding the polypeptide.Variants of recombinant antibodies may also be constructed byrestriction fragment manipulation or by overlap extension PCR withsynthetic oligonucleotides. ADCs of Formula (I) include, but are notlimited to, antibodies that have 1, 2, 3, or 4 engineered cysteine aminoacids (Lyon et al. (2012) Methods Enzymol. 502:123-38). In someembodiments, one or more free cysteine residues are already present inan antibody or antigen binding fragment, without the use of engineering,in which case the existing free cysteine residues may be used toconjugate the antibody or antigen binding fragment to a drug moiety.

Where more than one nucleophilic group reacts with a drug-linkerintermediate or a linker moiety reagent followed by drug moiety reagent,in a reaction mixture comprising multiple copies of the antibody orantigen binding fragment and linker moiety, then the resulting productcan be a mixture of ADC compounds with a distribution of one or moredrug moieties attached to each copy of the antibody or antigen bindingfragment in the mixture. In some embodiments, the drug loading in amixture of ADCs resulting from a conjugation reaction ranges from 1 to10 drug moieties attached per antibody or antigen binding fragment. Theaverage number of drug moieties per antibody or antigen binding fragment(i.e., the average drug loading, or average p) may be calculated by anyconventional method known in the art, e.g., by mass spectrometry (e.g.,reverse-phase LC-MS), and/or high-performance liquid chromatography(e.g., HIC-HPLC). In some embodiments, the average number of drugmoieties per antibody or antigen binding fragment is determined byhydrophobic interaction chromatography-high performance liquidchromatography (HIC-HPLC). In some embodiments, the average number ofdrug moieties per antibody or antigen binding fragment is determined byreverse-phase liquid chromatography-mass spectrometry (LC-MS). In someembodiments, the average number of drug moieties per antibody or antigenbinding fragment is from about 1.5 to about 3.5, about 2.5 to about 4.5,about 3.5 to about 5.5, about 4.5 to about 6.5, about 5.5 to about 7.5,about 6.5 to about 8.5, or about 7.5 to about 9.5. In some embodiments,the average number of drug moieties per antibody or antigen bindingfragment is from about 2 to about 4, about 3 to about 5, about 4 toabout 6, about 5 to about 7, about 6 to about 8, about 7 to about 9,about 2 to about 8, or about 4 to about 8.

In some embodiments, the average number of drug moieties per antibody orantigen binding fragment is about 2. In some embodiments, the averagenumber of drug moieties per antibody or antigen binding fragment isabout 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In some embodiments,the average number of drug moieties per antibody or antigen bindingfragment is 2.

In some embodiments, the average number of drug moieties per antibody orantigen binding fragment is about 4. In some embodiments, the averagenumber of drug moieties per antibody or antigen binding fragment isabout 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about4.1, about 4.2, about 4.3, about 4.4, or about 4.5. In some embodiments,the average number of drug moieties per antibody or antigen bindingfragment is 4.

In some embodiments, the average number of drug moieties per antibody orantigen binding fragment is about 8. In some embodiments, the averagenumber of drug moieties per antibody or antigen binding fragment isabout 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about8.1, about 8.2, about 8.3, about 8.4, or about 8.5. In some embodiments,the average number of drug moieties per antibody or antigen bindingfragment is 8.

In various embodiments, the term “about,” as used with respect to theaverage number of drug moieties per antibody or antigen bindingfragment, means plus or minus 10%.

Individual ADC compounds, or “species,” may be identified in the mixtureby mass spectroscopy and separated by UPLC or HPLC, e.g. hydrophobicinteraction chromatography (HIC-HPLC). In certain embodiments, ahomogeneous or nearly homogenous ADC product with a single loading valuemay be isolated from the conjugation mixture, e.g., by electrophoresisor chromatography.

In some embodiments, higher drug loading (e.g., p>8) may causeaggregation, insolubility, toxicity, or loss of cellular permeability ofcertain antibody-drug conjugates. Higher drug loading may alsonegatively affect the pharmacokinetics (e.g., clearance) of certainADCs. In some embodiments, lower drug loading (e.g., p<2) may reduce thepotency of certain ADCs against target-expressing cells and/or bystandercells. In some embodiments, the drug loading for an ADC of the presentdisclosure ranges from about 2 to about 8; from about 2 to about 6; fromabout 2 to about 5; from about 3 to about 5; from about 2 to about 4; orfrom about 4 to about 8.

In some embodiments, a drug loading and/or an average drug loading ofabout 2 is achieved, e.g., using partial reduction of intrachaindisulfides on the antibody or antigen binding fragment, and providesbeneficial properties. In some embodiments, a drug loading and/or anaverage drug loading of about 4 is achieved, e.g., using partialreduction of intrachain disulfides on the antibody or antigen bindingfragment, and provides beneficial properties. In some embodiments, adrug loading and/or an average drug loading of about 8 is achieved,e.g., using partial reduction of intrachain disulfides on the antibodyor antigen binding fragment, and provides beneficial properties. In someembodiments, a drug loading and/or an average drug loading of less thanabout 2 may result in an unacceptably high level of unconjugatedantibody species, which can compete with the ADC for binding to a targetantigen and/or provide for reduced treatment efficacy. In someembodiments, a drug loading and/or average drug loading of more thanabout 8 may result in an unacceptably high level of productheterogeneity and/or ADC aggregation. A drug loading and/or an averagedrug loading of more than about 8 may also affect stability of the ADC,due to loss of one or more chemical bonds required to stabilize theantibody or antigen binding fragment.

The present disclosure includes methods of producing the described ADCs.Briefly, the ADCs comprise an antibody or antigen binding fragment asthe antibody or antigen binding fragment, a drug moiety (e.g., asplicing modulator), and a linker that joins the drug moiety and theantibody or antigen binding fragment. In some embodiments, the ADCs canbe prepared using a linker having reactive functionalities forcovalently attaching to the drug moiety and to the antibody or antigenbinding fragment. For example, in some embodiments, a cysteine thiol ofan antibody or antigen binding fragment can form a bond with a reactivefunctional group of a linker or a drug-linker intermediate (e.g., amaleimide moiety) to make an ADC. The generation of the ADCs can beaccomplished by any technique known to the skilled artisan.

In some embodiments, an ADC is produced by contacting an antibody orantigen binding fragment with a linker and a drug moiety (e.g., asplicing modulator) in a sequential manner, such that the antibody orantigen binding fragment is covalently linked to the linker first, andthen the pre-formed antibody-linker intermediate reacts with the drugmoiety. The antibody-linker intermediate may or may not be subjected toa purification step prior to contacting the drug moiety. In otherembodiments, an ADC is produced by contacting an antibody or antigenbinding fragment with a linker-drug compound pre-formed by reacting alinker with a drug moiety. The pre-formed linker-drug compound may ormay not be subjected to a purification step prior to contacting theantibody or antigen binding fragment. In other embodiments, the antibodyor antigen binding fragment contacts the linker and the drug moiety inone reaction mixture, allowing simultaneous formation of the covalentbonds between the antibody or antigen binding fragment and the linker,and between the linker and the drug moiety. This method of producingADCs may include a reaction, wherein the antibody or antigen bindingfragment contacts the antibody or antigen binding fragment prior to theaddition of the linker to the reaction mixture, and vice versa. Incertain embodiments, an ADC is produced by reacting an antibody orantigen binding fragment with a linker joined to a drug moiety, such asADL1-splicing modulator (e.g., ADL1-D1) or ADL5-splicing modulator(e.g., ADL5-D2), under conditions that allow conjugation.

The ADCs prepared according to the methods described above may besubjected to a purification step. The purification step may involve anybiochemical methods known in the art for purifying proteins, or anycombination of methods thereof. These include, but are not limited to,tangential flow filtration (TFF), affinity chromatography, ion exchangechromatography, any charge or isoelectric point-based chromatography,mixed mode chromatography, e.g., CHT (ceramic hydroxyapatite),hydrophobic interaction chromatography, size exclusion chromatography,dialysis, filtration, selective precipitation, or any combinationthereof.

Therapeutic Uses and Compositions

Disclosed herein are methods of using the disclosed ADCs andcompositions in treating a subject for a disorder, e.g., a neoplasticdisorder. ADCs may be administered alone or in combination with a secondtherapeutic agent, and may be administered in any pharmaceuticallyacceptable formulation, dosage, and dosing regimen. ADC treatmentefficacy may be evaluated for toxicity as well as indicators of efficacyand adjusted accordingly. Efficacy measures include, but are not limitedto, a cytostatic and/or cytotoxic effect observed in vitro or in vivo,reduced tumor volume, tumor growth inhibition, and/or prolongedsurvival.

Methods of determining whether an ADC exerts a cytostatic and/orcytotoxic effect on a cell are known. For example, the cytotoxic orcytostatic activity of an ADC can be measured by: exposing mammaliancells expressing a target protein of the ADC in a cell culture medium;culturing the cells for a period from about 6 hours to about 6 days; andmeasuring cell viability. Cell-based in vitro assays may also be used tomeasure viability (proliferation), cytotoxicity, and induction ofapoptosis (caspase activation) of the ADC.

For determining whether an ADC exerts a cytostatic effect, a thymidineincorporation assay may be used. For example, cancer cells expressing atarget antigen at a density of 5,000 cells/well of a 96-well plated canbe cultured for a 72-hour period and exposed to 0.5 μCi of ³H-thymidineduring the final 8 hours of the 72-hour period. The incorporation of³H-thymidine into cells of the culture is measured in the presence andabsence of the ADC.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) may be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis can be quantitated, for example, bymeasuring DNA fragmentation. Commercial photometric methods for thequantitative in vitro determination of DNA fragmentation are available.Examples of such assays, including TUNEL (which detects incorporation oflabeled nucleotides in fragmented DNA) and ELISA-based assays, aredescribed in Biochemica (1999) No. 2, pp. 34-37 (Roche MolecularBiochemicals).

Apoptosis may also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal., eds. (1992) pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Apoptosis may also be determined, in someembodiments, by screening for caspase activity. In some embodiments, aCaspase-Glo® Assay can be used to measure activity of caspase-3 andcaspase-7. In some embodiments, the assay provides a luminogeniccaspase-3/7 substrate in a reagent optimized for caspase activity,luciferase activity, and cell lysis. In some embodiments, addingCaspase-Glo® 3/7 Reagent in an “add-mix-measure” format may result incell lysis, followed by caspase cleavage of the substrate and generationof a “glow-type” luminescent signal, produced by luciferase. In someembodiments, luminescence may be proportional to the amount of caspaseactivity present, and can serve as an indicator of apoptosis. Othermorphological changes that can be measured to determine apoptosisinclude, e.g., cytoplasmic condensation, increased membrane blebbing,and cellular shrinkage. Determination of any of these effects on cancercells indicates that an ADC is useful in the treatment of cancers.

Cell viability may be measured, e.g., by determining in a cell theuptake of a dye such as neutral red, trypan blue, Crystal Violet, orALAMAR™ blue (see, e.g., Page et al. (1993) Intl J Oncology 3:473-6). Insuch an assay, the cells are incubated in media containing the dye, thecells are washed, and the remaining dye, reflecting cellular uptake ofthe dye, is measured spectrophotometrically. Cell viability may also bemeasured, e.g., by quantifying ATP, an indicator of metabolically activecells. In certain embodiments, in vitro potency and/or cell viability ofprepared ADCs or splicing modulator compounds may be assessed using aCellTiter-Glo® Luminescent Cell Viability Assay, as described in theexamples provided herein. In this assay, in certain embodiments, thesingle reagent (CellTiter-Glo® Reagent) is added directly to cellscultured in serum-supplemented medium. The addition of reagent resultsin cell lysis and generation of a luminescent signal proportional to theamount of ATP present. The amount of ATP is directly proportional to thenumber of cells present in culture. The protein-binding dyesulforhodamine B (SRB) can also be used to measure cytotoxicity (Skehanet al. (1990) J Natl Cancer Inst. 82:1107-12).

The disclosed ADCs may also be evaluated for bystander killing activity.Bystander killing activity may be determined, e.g., by an assayemploying two cell lines, one positive for a target antigen and onenegative for a target antigen. In certain embodiments, the design of theassay allows tracking of only target negative cells. In certainembodiments, cells are plated under three conditions: (i) targetnegative cells alone (tagged or labeled); (ii) target positive cellsalone; and (iii) co-culture of target negative cells and target positivecells. Cells are then treated with an ADC followed by monitoring ofcytotoxicity. When plates are read with CellTiter-Glo® Reagent,viability of all cell populations can be monitored. When plates are readwith OneGlo® Reagent, only the tagged or labeled target negative cellsproduce a signal. Killing of the target-negative cells when mixed withtarget-positive cells is indicative of bystander killing, whereaskilling of the target-negative cells in the absence of thetarget-positive cells is indicative of off-target killing.

In certain aspects, the present disclosure features a method of killing,inhibiting or modulating the growth of, or interfering with themetabolism of, a cancer cell or tissue by disrupting RNA splicing. Themethod may be used with any subject where disruption of RNA splicingprovides a therapeutic benefit. Subjects that may benefit fromdisrupting RNA splicing include, but are not limited to, those having orat risk of having a neoplastic disorder such as a hematologicalmalignancy or a solid tumor. In certain embodiments, the hematologicalmalignancy is a B-cell malignancy, a cancer of the blood (leukemia), acancer of plasma cells (myeloma, e.g., multiple myeloma), or a cancer ofthe lymph nodes (lymphoma). In certain embodiments, the hematologicalmalignancy is acute myelogenous leukemia or multiple myeloma. In certainembodiments, the leukemia is acute lymphoblastic leukemia (ALL), acutemyelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronicmyelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), oracute monocytic leukemia (AMoL). In certain embodiments, the lymphoma isHodgkin's lymphoma or non-Hodgkin's lymphoma. In certain embodiments,the hematological malignancy is myelodysplasia syndrome (MDS). Incertain embodiments, the solid tumor is a carcinoma such as breastcancer, pancreatic cancer, prostate cancer, colon or colorectal cancer,lung cancer, gastric cancer, cervical cancer, endometrial cancer,ovarian cancer, cholangiocarcinoma, glioma, or melanoma. In certainembodiments, the solid tumor is breast cancer, gastric cancer, prostatecancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterinecancer (e.g., uterine serous endometrial carcinoma), salivary ductcarcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer,kidney cancer, colorectal cancer, and esophageal cancer. In certainembodiments, the lung cancer is lung adenocarcinoma. In certainembodiments, the uterine cancer is uterine serous endometrial carcinoma.

In various embodiments, the disclosed ADCs may be administered in anycell or tissue that expresses HER2, such as a HER2-expressing neoplasticcell or tissue. An exemplary embodiment includes a method of inhibitingHER2-mediated cell signaling or a method of killing a cell. The methodmay be used with any cell or tissue that expresses HER2, such as acancerous cell or a metastatic lesion. Non-limiting examples ofHER2-expressing cancers include breast cancer, gastric cancer, bladdercancer, urothelial cell carcinoma, esophageal cancer, lung cancer (e.g.,lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrialcarcinoma), salivary duct carcinoma, cervical cancer, endometrialcancer, and ovarian cancer (English et al. (2013) Mol Diagn Ther.17:85-99). Non-limiting examples of HER2-expressing cells includeHCC1954 and SKBR3 human breast ductal carcinoma cells, N87 human gastriccarcinoma cells, and cells comprising a recombinant nucleic acidencoding HER2 or a portion thereof.

In various embodiments, the disclosed ADCs may be administered in anycell or tissue that expresses CD138, such as a CD138-expressingneoplastic cell or tissue. An exemplary embodiment includes a method ofinhibiting CD138-mediated cell signaling or a method of killing a cell.The method may be used with any cell or tissue that expresses CD138,such as a cancerous cell or a metastatic lesion. Non-limiting examplesof CD138-expressing cancers include intrathoracic cancer (e.g., lungcancer, mesothelioma), skin cancer (e.g., basal cell carcinoma, squamouscell carcinoma), head and neck cancer (e.g., laryngeal, hypopharynx,nasopharyngeal), breast cancer, urogenital cancer (e.g., cervicalcancer, ovarian cancer, endometrial cancer, prostate cancer, bladdercancer, urothelial cancer), hematological malignancies (e.g., myelomasuch as multiple myeloma, Hodgkin's lymphoma), and thyroid cancer(Szatmári et al. (2015) Dis Markers 2015:796052). Non-limiting examplesof CD138-expressing cells include MOLP8 human multiple myeloma cells,and cells comprising a recombinant nucleic acid encoding CD138 or aportion thereof.

In various embodiments, the disclosed ADCs may be administered in anycell or tissue that expresses EPHA2, such as an EPHA2-expressingneoplastic cell or tissue. An exemplary embodiment includes a method ofinhibiting EPHA2-mediated cell signaling or a method of killing a cell.The method may be used with any cell or tissue that expresses EPHA2,such as a cancerous cell or a metastatic lesion. Non-limiting examplesof EPHA2-expressing cancers include breast cancer, brain cancer, ovariancancer, bladder cancer, pancreatic cancer, esophageal cancer, lungcancer, prostate cancer, melanoma, esophageal cancer, and gastric cancer(Tandon et al. (2011) Expert Opin Ther Targets 15(1):31-51. Non-limitingexamples of EPHA2-expressing cells include PC3 human prostate cancercells, and cells comprising a recombinant nucleic acid encoding EPHA2 ora portion thereof.

Exemplary methods include the steps of contacting a cell with an ADC, asdescribed herein, in an effective amount, i.e., amount sufficient tokill the cell. The method can be used on cells in culture, e.g. invitro, in vivo, ex vivo, or in situ. For example, cells that expressHER2 (e.g., cells collected by biopsy of a tumor or metastatic lesion;cells from an established cancer cell line; or recombinant cells), canbe cultured in vitro in culture medium and the contacting step can beaffected by adding the ADC to the culture medium. The method will resultin killing of cells expressing HER2, including in particular tumor cellsexpressing HER2. Alternatively, the ADC can be administered to a subjectby any suitable administration route (e.g., intravenous, subcutaneous,or direct contact with a tumor tissue) to have an effect in vivo. Thisapproach can be used for antibodies targeting other cell surfaceantigens (e.g., CD138, EPHA2).

The in vivo effect of a disclosed ADC therapeutic composition can beevaluated in a suitable animal model. For example, xenogeneic cancermodels can be used, wherein cancer explants or passaged xenografttissues are introduced into immune compromised animals, such as nude orSCID mice (Klein et al. (1997) Nature Med. 3:402-8). Efficacy may bepredicted using assays that measure inhibition of tumor formation, tumorregression or metastasis, and the like.

In vivo assays that evaluate the promotion of tumor death by mechanismssuch as apoptosis may also be used. In one embodiment, xenografts fromtumor bearing mice treated with the therapeutic composition can beexamined for the presence of apoptotic foci and compared to untreatedcontrol xenograft-bearing mice. The extent to which apoptotic foci arefound in the tumors of the treated mice provides an indication of thetherapeutic efficacy of the composition.

Further provided herein are methods of treating a neoplastic disorder,e.g., a cancer. The ADCs disclosed herein can be administered to anon-human mammal or human subject for therapeutic purposes. Thetherapeutic methods entail administering to a subject having orsuspected of having a neoplastic disorder a therapeutically effectiveamount of an ADC or composition comprising a splicing modulator linkedto a targeting antibody that binds to an antigen expressed, isaccessible to binding, or is localized on a cancer cell surface. In someembodiments, treatment with the antibody-drug conjugate or compositioninduces bystander killing of neoplastic cells which do not express atarget antigen but are adjacent to neoplastic cells which express atarget antigen.

An exemplary embodiment is a method of delivering a splicing modulatorto a cell expressing HER2, comprising conjugating the splicing modulatorto an antibody that immunospecifically binds to a HER2 epitope andexposing the cell to the ADC. Exemplary tumor cells that express HER2for which the ADCs of the present disclosure are indicated includegastric carcinoma cells and breast ductal carcinoma cells.

Another exemplary embodiment is a method of delivering a splicingmodulator to a cell expressing CD138, comprising conjugating thesplicing modulator to an antibody that immunospecifically binds to aCD138 epitope and exposing the cell to the ADC. Exemplary tumor cellsthat express CD138 for which the ADCs of the present disclosure areindicated include multiple myeloma cells.

Another exemplary embodiment is a method of delivering a splicingmodulator to a cell expressing EPHA2, comprising conjugating thesplicing modulator to an antibody that immunospecifically binds to anEPHA2 epitope and exposing the cell to the ADC. Exemplary tumor cellsthat express EPHA2 for which the ADCs of the present disclosure areindicated include prostate cancer cells.

Another exemplary embodiment is a method of reducing or inhibitinggrowth of a tumor (e.g., a HER2-expressing tumor, a CD138-expressingtumor, an EPHA2-expressing tumor), comprising administering atherapeutically effective amount of an ADC or composition comprising anADC. In some embodiments, the treatment is sufficient to reduce orinhibit the growth of the patient's tumor, reduce the number or size ofmetastatic lesions, reduce tumor load, reduce primary tumor load, reduceinvasiveness, prolong survival time, and/or maintain or improve thequality of life. In some embodiments, the tumor is resistant orrefractory to treatment with the antibody or antigen binding fragment ofthe ADC (e.g., an anti-HER2 antibody, an anti-CD138 antibody, ananti-EPHA2 antibody) when administered alone, and/or the tumor isresistant or refractory to treatment with the splicing modulator drugmoiety when administered alone

In certain aspects, the present disclosure provides a method of reducingor inhibiting growth of a HER2-expressing tumor. In certain embodiments,treatment with the antibody-drug conjugate or composition inducesbystander killing of tumor cells which do not express HER2 but that areadjacent to neoplastic tumor cells which do express HER2. ExemplaryHER2-expressing tumor types include but are not limited to tumorsderived from a HER2-expressing breast cancer, gastric cancer, bladdercancer, urothelial cell carcinoma, esophageal cancer, lung cancer (e.g.,lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrialcarcinoma), salivary duct carcinoma, cervical cancer, endometrialcancer, and ovarian cancer. In certain embodiments, the HER2-expressingtumor is a tumor derived from a HER2-expressing breast cancer, ovariancancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterinecancer (e.g., uterine serous endometrial carcinoma), osteosarcoma, orsalivary duct carcinoma. In certain embodiments, the HER2-expressingtumor is a lung adenocarcinoma or uterine serous endometrial carcinoma.

In certain aspects, the present disclosure provides a method of reducingor inhibiting growth of a CD138-expressing tumor. In certainembodiments, treatment with the antibody-drug conjugate or compositioninduces bystander killing of tumor cells which do not express CD138 butthat are adjacent to neoplastic tumor cells which do express CD138.Exemplary CD138-expressing tumor types include but are not limited totumors derived from a CD138-expressing intrathoracic cancer (e.g., lungcancer, mesothelioma), skin cancer (e.g., basal cell carcinoma, squamouscell carcinoma), head and neck cancer (e.g., laryngeal, hypopharynx,nasopharyngeal), breast cancer, urogenital cancer (e.g., cervicalcancer, ovarian cancer, endometrial cancer, prostate cancer, bladdercancer, urothelial cancer), and thyroid cancer.

In certain aspects, the present disclosure provides a method of reducingor inhibiting growth of an EPHA2-expressing tumor. In certainembodiments, treatment with the antibody-drug conjugate or compositioninduces bystander killing of tumor cells which do not express EPHA2 butthat are adjacent to neoplastic tumor cells which do express EPHA2.Exemplary EPHA2-expressing tumor types include but are not limited totumors derived from an EPHA2-expressing breast cancer, brain cancer,ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer,lung cancer, prostate cancer, melanoma, esophageal cancer, and gastriccancer. In certain embodiments, the EPHA2-expressing tumor is a tumorderived from an EPHA2-expressing breast cancer, prostate cancer, ovariancancer, lung cancer, melanoma, colon cancer, or esophageal cancer.

Moreover, antibodies of the present disclosure may be administered to anon-human mammal expressing an antigen with which the ADC is capable ofbinding for veterinary purposes or as an animal model of human disease.Regarding the latter, such animal models may be useful for evaluatingthe therapeutic efficacy of the disclosed ADCs (e.g., testing of dosagesand time courses of administration).

Further provided herein are therapeutic uses of the disclosed ADCs andcompositions. An exemplary embodiment is the use of an ADC in thetreatment of a neoplastic disorder (e.g., a HER2-expressing cancer, aCD138-expressing cancer, an EPHA2-expressing cancer). Another exemplaryembodiment is an ADC for use in the treatment of a neoplastic disorder(e.g., a HER2-expressing cancer, a CD138-expressing cancer, anEPHA2-expressing cancer). Methods for identifying subjects havingcancers that express a target antigen (e.g., HER2, CD138, EPHA2, MSLN,FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16,SLC39A6, SLC44A4, STEAP1) are known in the art and may be used toidentify suitable patients for treatment with a disclosed ADC.

Another exemplary embodiment is the use of an ADC in a method ofmanufacturing a medicament for the treatment of a neoplastic disorder(e.g., a HER2-expressing cancer, a CD138-expressing cancer, anEPHA2-expressing cancer).

The therapeutic compositions used in the practice of the foregoingmethods may be formulated into pharmaceutical compositions comprising apharmaceutically acceptable carrier suitable for the desired deliverymethod. An exemplary embodiment is a pharmaceutical compositioncomprising an ADC of the present disclosure and a pharmaceuticallyacceptable carrier. Suitable carriers include any material that, whencombined with the therapeutic composition, retains the anti-tumorfunction of the therapeutic composition and is generally non-reactivewith the patient's immune system. Pharmaceutically acceptable carriersinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike that are physiologically compatible. Examples of pharmaceuticallyacceptable carriers include one or more of water, saline, phosphatebuffered saline, dextrose, glycerol, ethanol, mesylate salt, and thelike, as well as combinations thereof. In many cases, isotonic agentsare included, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the ADC.

Therapeutic formulations may be solubilized and administered via anyroute capable of delivering the therapeutic composition to the tumorsite. Potentially effective routes of administration include, but arenot limited to, intravenous, parenteral, intraperitoneal, intramuscular,intratumor, intradermal, intraorgan, orthotopic, and the like.Therapeutic protein preparations can be lyophilized and stored assterile powders, e.g., under vacuum, and then reconstituted inbacteriostatic water (containing for example, benzyl alcoholpreservative) or in sterile water prior to injection. Therapeuticformulations may comprise an ADC or a pharmaceutically acceptable saltthereof, e.g., a mesylate salt.

In some embodiments, the ADC is administered to the patient daily,bimonthly, or any time period in between. Dosages and administrationprotocols for the treatment of cancers using the foregoing methods willvary with the method and the target cancer, and will generally depend ona number of other factors appreciated in the art.

Various delivery systems are known and may be used to administer one ormore ADCs of the present disclosure. Methods of administering the ADCsinclude, but are not limited to, parenteral administration (e.g.,intradermal, intramuscular, intraperitoneal, intravenous andsubcutaneous), epidural administration, intratumoral administration, andmucosal administration (e.g., intranasal and oral routes). In addition,pulmonary administration may be employed, e.g., by use of an inhaler ornebulizer, and formulation with an aerosolizing agent. See, e.g., thecompositions and methods for pulmonary administration described in U.S.Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064,5,855,913, 5,290,540, and 4,880,078; and Intl. Publ. Nos. WO1992/019244, WO 1997/032572, WO 1997/044013, WO 1998/031346, and WO1999/066903. The ADCs may be administered by any convenient route, forexample, by infusion or bolus injection, or by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.). Administration can be either systemic orlocal.

Therapeutic compositions disclosed herein may be sterile and stableunder the conditions of manufacture and storage. In some embodiments,one or more of the ADCs, or pharmaceutical compositions, is supplied asa dry sterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted (e.g., with wateror saline) to the appropriate concentration for administration to asubject. In some embodiments, one or more of the prophylactic ortherapeutic agents or pharmaceutical compositions is supplied as a drysterile lyophilized powder in a hermetically sealed container at a unitdosage of at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg,at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or atleast 100 mg, or any amount in between. In some embodiments, thelyophilized ADCs or pharmaceutical compositions is stored at between 2°C. and 8° C. in the original container. In some embodiments, one or moreof the ADCs or pharmaceutical compositions described herein is suppliedin liquid form in a hermetically sealed container, e.g., a containerindicating the quantity and concentration of the agent. In someembodiments, the liquid form of the administered composition is suppliedin a hermetically sealed container of at least 0.25 mg/mL, at least 0.5mg/mL, at least 1 mg/mL, at least 2.5 mg/mL, at least 5 mg/mL, at least8 mg/mL, at least 10 mg/mL, at least 15 mg/mL, at least 25 mg/mL, atleast 50 mg/mL, at least 75 mg/mL, or at least 100 mg/mL ADC. The liquidform may be stored at between 2° C. and 8° C. in the original container.

In some embodiments, the disclosed ADCs can be incorporated into apharmaceutical composition suitable for parenteral administration. Theinjectable solution may be composed of either a liquid or lyophilizeddosage form in a flint or amber vial, ampule, or pre-filled syringe, orother known delivery or storage device.

The compositions described herein may be in a variety of forms. Theseinclude, for example, liquid, semi-solid, and solid dosage forms, suchas liquid solutions (e.g., injectable and infusible solutions),dispersions or suspensions, tablets, pills, powders, liposomes, andsuppositories. The preferred form depends on the intended mode ofadministration and therapeutic application.

In various embodiments, treatment involves single bolus or repeatedadministration of the ADC preparation via an acceptable route ofadministration.

Patients may be evaluated for the levels of target antigen in a givensample (e.g. the levels of target antigen expressing cells) in order toassist in determining the most effective dosing regimen, etc. Anexemplary embodiment is a method of determining whether a patient willbe responsive to treatment with an ADC of the present disclosure,comprising providing a biological sample from the patient and contactingthe biological sample with the ADC. Exemplary biological samples includetissue or body fluid, such as an inflammatory exudate, blood, serum,bowel fluid, stool sample, or tumor biopsy (e.g., a tumor biopsy derivedfrom a patient having or at risk of a target antigen-expressing cancer,e.g., a HER2-expressing cancer, a CD138-expressing cancer, anEPHA2-expressing cancer). In some embodiments, a sample (e.g., a tissueand/or body fluid) can be obtained from a subject, and a suitableimmunological method can be used to detect and/or measure proteinexpression of the target antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1,CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6,SLC44A4, STEAP1). Such evaluations are also used for monitoring purposesthroughout therapy, and are useful to gauge therapeutic success incombination with the evaluation of other parameters.

In some embodiments, the efficacy of an ADC may be evaluated bycontacting a tumor sample from a subject with the ADC and evaluatingtumor growth rate or volume. In some embodiments, when an ADC has beendetermined to be effective, it may be administered to the subject.

The above therapeutic approaches can be combined with any one of a widevariety of additional surgical, chemotherapy, or radiation therapyregimens. In some embodiments, the ADCs or compositions disclosed hereinare co-formulated and/or co-administered with one or more additionaltherapeutic agents, e.g., one or more chemotherapeutic agents.Non-limiting examples of chemotherapeutic agents include alkylatingagents, for example, nitrogen mustards, ethyleneimine compounds, andalkyl sulphonates; antimetabolites, for example, folic acid, purine orpyrimidine antagonists; anti-mitotic agents, for example, anti-tubulinagents such as eribulin or eribulin mesylate (Halaven™), vincaalkaloids, and auristatins; cytotoxic antibiotics; compounds that damageor interfere with DNA expression or replication, for example, DNA minorgroove binders; and growth factor receptor antagonists. In someembodiments, a chemotherapeutic agent may be a cytotoxic or cytostaticagent. Examples of cytotoxic agents include, but are not limited to,anti-mitotic agents, such as eribulin or eribulin mesylate (Halaven™),auristatins (e.g., monomethyl auristatin E (MMAE), monomethyl auristatinF (MMAF)), maytansinoids (e.g., maytansine), dolastatins, duostatins,cryptophycins, vinca alkaloids (e.g., vincristine, vinblastine),taxanes, taxols, and colchicines; anthracyclines (e.g., daunorubicin,doxorubicin, dihydroxyanthracindione); cytotoxic antibiotics (e.g.,mitomycins, actinomycins, duocarmycins (e.g., CC-1065), auromycins,duomycins, calicheamicins, endomycins, phenomycins); alkylating agents(e.g., cisplatin); intercalating agents (e.g., ethidium bromide);topoisomerase inhibitors (e.g., etoposide, tenoposide); radioisotopes,such as At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹² or ²¹³, P³²,and radioactive isotopes of lutetium (e.g., Lu¹⁷⁷); and toxins ofbacterial, fungal, plant or animal origin (e.g., ricin (e.g., ricinA-chain), diphtheria toxin, Pseudomonas exotoxin A (e.g., PE40),endotoxin, mitogellin, combrestatin, restrictocin, gelonin,alpha-sarcin, abrin (e.g., abrin A-chain), modeccin (e.g., modeccinA-chain), curicin, crotin, Sapaonaria officinalis inhibitor,glucocorticoid).

Also disclosed herein are uses of one or more of the disclosed ADCs inthe manufacture of a medicament for treating cancer, e.g., according tothe methods described above. In some embodiments, the ADCs disclosedherein are used for treating cancer, e.g., according to the methodsdescribed above.

In various embodiments, kits for use in the laboratory and therapeuticapplications described herein are within the scope of the presentdisclosure. Such kits may comprise a carrier, package, or container thatis compartmentalized to receive one or more containers such as vials,tubes, and the like, each of the container(s) comprising one of theseparate elements to be used in a method disclosed herein, along with alabel or insert comprising instructions for use, such as a use describedherein. Kits may comprise a container comprising a drug moiety. Thepresent disclosure also provides one or more of the ADCs, orpharmaceutical compositions thereof, packaged in a hermetically sealedcontainer, such as an ampoule or sachette, indicating the quantity ofthe agent.

Kits may comprise the container described above, and one or more othercontainers associated therewith that comprise materials desirable from acommercial and user standpoint, including buffers, diluents, filters,needles, syringes; carrier, package, container, vial and/or tube labelslisting contents and/or instructions for use, and package inserts withinstructions for use.

A label may be present on or with the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, such as a prognostic, prophylactic, diagnostic, orlaboratory application. A label may also indicate directions for eitherin vivo or in vitro use, such as those described herein. Directions andor other information may also be included on an insert(s) or label(s),which is included with or on the kit. The label may be on or associatedwith the container. A label may be on a container when letters, numbers,or other characters forming the label are molded or etched into thecontainer itself. A label may be associated with a container when it ispresent within a receptacle or carrier that also holds the container,e.g., as a package insert. The label may indicate that the compositionis used for diagnosing or treating a condition, such as a cancer adescribed herein.

Neoantigens and Methods of Use

Also disclosed herein, in various embodiments, are methods of treating apatient by inducing neoantigens in tumor cells that can be targeted bythe patient's immune system for clearance. Without being bound bytheory, in various embodiments, administering a splicing modulator,alone and/or as part of an ADC or composition, may produce neoantigensthat induce an immune response, induce a double-stranded RNA immuneresponse, e.g., as a result of re-expressed intron-resident endogenousretroviruses, and/or produce neoantigens that induce immunogenic celldeath.

As used herein, the term “neoantigen” refers to any antigen to which theimmune system has not previously been exposed that arises from one ormore tumor-specific mutations and/or from exposing a tumor to a drug(e.g., any one or more of the splicing modulators disclosed herein,alone and/or as part of an ADC or composition). Tumor-specific mutationscan include missense mutations, frameshifts, translocations, and mRNAsplicing variants, as well as mutations that influence posttranslationalprocessing, such as phosphorylation and glycosylation. These exemplarymutations, in various embodiments, can be derived from non-synonymouscoding changes and/or mutations that alter mRNA processing (e.g.,splicing). All of these exemplary mutations, in various embodiments, canresult in molecular changes that can be discriminated by an appropriateT-cell receptor. In various preferred embodiments, an exemplaryneoantigen is a neoantigen induced by delivery of a splicing modulator,alone and/or as part of an ADC or composition. In various embodiments,delivery of a splicing modulator (e.g., any one or more of the splicingmodulators disclosed herein) can induce novel mRNA splicing that resultsin the translation of proteins containing one or more novel peptidedomains to which the immune system has not previously been exposed. Invarious embodiments, tumor-specific mutations may be mRNA splicingvariants resulting from delivery or administration of a splicingmodulator, ADC, or composition comprising a splicing modulator or ADC.

Without being bound by theory, in various embodiments, the delivery ofsplicing modulators, alone and as part of an ADC or composition, mayinduce novel mRNA splicing (e.g., exon skipping, intron retention) thatresults in the alteration of the open reading frames and/or codingsequences of various genes. In various embodiments, these altered genesare translated into proteins containing one or more novel peptidedomains recognized by the immune system as foreign. In variousembodiments, the one or more novel peptide domains do not exist in theproteins or in any other part of the human proteome in the absence ofsplicing modulator treatment. In various embodiments, the proteinscontaining the one or more novel peptide domains can be degraded by theproteasome to create novel peptide fragments that act as substrates forthe immunopeptide presentation machinery, e.g., via MHC presentation. Invarious embodiments, the novel peptide fragments representingneoantigens can be presented in the MHC1-bound peptidome, e.g., on tumorcells.

In various embodiments, the delivery of splicing modulators, alone andas part of an ADC or composition, may lead to one or more tumorcell-intrinsic events (e.g., cell growth arrest). In variousembodiments, the tumor cell-intrinsic event(s) may lead to (1) enhancedengagement by phagocytic cells (Bracci et al. (2014) Cell Death Differ.21(1):15-25); (2) the transport of novel peptide fragments to a tumordraining lymph node to engage with antigen-presenting cells; (3)antigen-presenting cells processing novel peptide fragments from aphagocytosed tumor cell and presenting the fragments as neoantigens tocirculating naïve T-cell populations; (4) novel peptide fragmentsinteracting with T-cells expressing receptors that recognize thefragments as neoantigens; (5) maturation and activation of effectorT-cell responses (e.g., CD4+ and/or CD8+ T-cells; and/or (6) engagementof T-cells with additional tumor cells exposed to the splicing modulatortreatment and presenting novel peptide fragments representingneoantigens on their surface MHC1 complexes. In various embodiments, thetumor cell-intrinsic event(s) may result, either directly or indirectly,in T-cell engagement of effector function and/or killing ofneoantigen-presenting tumor cells.

Also, without being bound by theory, in various embodiments, thedelivery of splicing modulators, alone and as part of an ADC orcomposition, may cause the re-expression of intron-resident endogenousretroviruses, leading to a double-stranded RNA immune response.

Further, without being bound by theory, in various embodiments, thedelivery of splicing modulators, alone and as part of an ADC orcomposition, may lead to immunogenic cell death triggered by splicemodulator-induced release of mutationally-derived neoantigens. Invarious embodiments, the delivery of splicing modulators, alone and aspart of an ADC or composition, may induce a double-stranded RNA immuneresponse. In various embodiments, the double-stranded RNA immuneresponse can result from the re-expression of intron-resident endogenousretroviruses. In various embodiments, the double-stranded RNA immuneresponse can result in tumor cell death. In various embodiments, thedelivery of splicing modulators, alone and as part of an ADC orcomposition, may induce immunogenic cell death. In various embodiments,the immunogenic cell death can result from release of mutational-derivedneoantigens and/or a host immune response against tumor cells.

Accordingly, in various embodiments, methods of treatment are disclosedcomprising inducing neoantigens by administering one or more splicingmodulators and/or ADCs and/or compositions comprising a splicingmodulator or ADC, e.g., any splicing modulator, ADC, or compositiondisclosed herein. In various embodiments, the method comprisesadministering a reduced dosage of the splicing modulator, ADC, orcomposition than would be needed absent the induction of neoantigens. Insome embodiments, the method comprises administering one or more initialinduction doses to produce neoantigens and induce an immune response(e.g., converting naïve T-cells to memory cells), followed by a reduceddosage or administration frequency (i.e., because of the combinatorialeffect of the splicing modulator, ADC, or composition and of immunetargeting of the neoantigens). In some embodiments, treatment cancomprise a combination of administering the splicing modulator, ADC, orcomposition to induce a neoantigen-based immune response and at leastone additional therapy (e.g., a second anti-cancer therapy). Forexample, in some embodiments, treatment can comprise a combination ofadministering the splicing modulator, ADC, or composition to induce aneoantigen-based immune response and one or more checkpoint inhibitors.In some embodiments, treatment can comprise a combination ofadministering the splicing modulator, ADC, or composition to induce aneoantigen-based immune response and one or more cytokines or cytokineanalogs. In some embodiments, treatment can comprise a combination ofadministering the splicing modulator, ADC, or composition to induce aneoantigen-based immune response and one or more neoantigen vaccines. Insome other embodiments, treatment can comprise a combination ofadministering the splicing modulator, ADC, or composition to induce aneoantigen-based immune response and one or more engineeredtumor-targeting T-cells (e.g., CAR-T).

In some embodiments, neoantigens can be used to monitor theeffectiveness of treatment with a splicing modulator, ADC, orcomposition. For instance, after administration of a splicing modulator,ADC, or composition, a patient sample (e.g., a tumor biopsy) can beobtained and screened for neoantigens or for identifiers of an immune orinflammatory response. Further treatment can be provided, e.g., atreduced dosage, if a neoantigen and/or immune response is detected.

In various embodiments, methods of treatment are disclosed comprisinginducing a double-stranded RNA immune response by administering one ormore splicing modulators and/or ADCs and/or compositions comprising asplicing modulator or ADC, e.g., any splicing modulator, ADC, orcomposition disclosed herein.

In various embodiments, methods of treatment are disclosed comprisinginducing immunogenic cell death by administering one or more splicingmodulators and/or ADCs and/or compositions comprising a splicingmodulator or ADC, e.g., any splicing modulator, ADC, or compositiondisclosed herein.

In various embodiments, administration of a splicing modulator, ADC, orcomposition comprising a splicing modulator can be combined with anyknown anti-cancer therapy. Examples of current immune activatingstrategies available for oncology treatment include, but are not limitedto, treatment with immune checkpoint inhibitor (ICI) molecules,treatment with cytokines or cytokine analogs, vaccination withtumor-associated vaccines, and engineering tumor-targeting T-cells(e.g., expansion of tumor-infiltrating lymphocytes or CAR-T). Thesetechnologies are predominantly focused on enhancing or inducing animmune response to already existing tumor antigens (either mutations oraberrant expression of cell-surface proteins). One or more of thesestrategies may involve one or more mutations that are capable ofinducing an antigenic T-cell response. For example, patient responses tocheckpoint inhibition may correlate with non-synonymous mutationalburden. In addition, cancer vaccine approaches may be used that rely onpre-existing mutations and the antigenicity of these mutations.

Splicing modulators and/or ADCs comprising such modulators may inducebroad-ranging changes in the transcriptome that occur in multiplelineages. Translation of these mRNA changes may produce robust andreproducible protein changes that produce MHC1-bound neopeptides withhigh affinity across multiple HLA isotypes. Without being bound bytheory, due to the large number of changes to the transcriptome andproteome, treatment with splicing modulators and/or ADCs may enrich thenumber of potentially reactive neoantigens for enhanced engagement ofthe adaptive immune response.

As described herein, the terms “splicing modulator,” “spliceosomemodulator,” or “splice modulator” refer to compounds that haveanti-cancer activity by interacting with components of the spliceosome.In some embodiments, a splicing modulator alters the rate or form ofsplicing in a target cell. Splicing modulators that function asinhibitory agents, for example, are capable of decreasing uncontrolledcellular proliferation. In particular, in some embodiments, the splicingmodulators may act by inhibiting the SF3b spliceosome complex. In someembodiments, a splicing modulator is chosen from any one or more of thesplicing modulators disclosed herein. In some embodiments, a splicingmodulator is used, delivered to a cell, and/or administered to a subjectas a stand-alone agent. In some other embodiments, a splicing modulatoris used, delivered to a cell, and/or administered to a subject as partof an ADC (e.g., an ADC chosen from any of the exemplary ADCs disclosedherein). In some other embodiments, a splicing modulator is used,delivered to a cell, and/or administered to a subject as part of acomposition comprising multiple copies of the splicing modulator ormultiple copies of an ADC carrying the splicing modulator. Suchcompositions are disclosed herein.

In some embodiments, a splicing modulator used, delivered to a cell,and/or administered to a subject as part of an ADC (e.g., an ADC chosenfrom any of the exemplary ADCs disclosed herein) provides addedtherapeutic benefits over a splicing modulator used, delivered to acell, and/or administered to a subject as a stand-alone agent. Forexample, in some embodiments, a splicing modulator used, delivered to acell, and/or administered to a subject as part of an ADC providestargeted delivery of the splicing modulator to a neoplastic cellexpressing the target antigen (i.e., the antigen targeted by theantibody moiety of the ADC). In some embodiments, such targeted deliveryof the splicing modulator reduces off-target treatment and/or off-targetcytotoxicity. In some embodiments, such targeted delivery promotestumor-selective neoantigen presentation on neoplastic cells, but not onhealthy cells that do not express the target antigen. In someembodiments, such targeted delivery leads to, e.g., at least 75%, 80%,85%, 90%, 95%, or 99%, of the alternative splicing and induction ofnovel mRNAs and MHC-associated peptides representing neoantigens intargeted neoplastic cells rather than off-target cells. Thus, withoutbeing bound by theory, in some embodiments, following effector T-cellpriming and/or expansion (e.g., using a neoantigen vaccine), the immunesystem may preferentially attack neoantigen-presenting neoplastic cellsrather than healthy cells due to the preferential expression ofneoantigens on tumor cells after treatment with an ADC as disclosedherein.

Immune Induction and Treatment Regimen:

In various embodiments, the present disclosure provides a method ofinducing at least one neoantigen by contacting a neoplastic cell with aneffective amount of a splicing modulator, a splicing modulator-basedantibody-drug conjugate (ADC), or a composition comprising a splicingmodulator or ADC. In various embodiments, the present disclosureprovides a method of inducing a double-stranded RNA immune response bycontacting a neoplastic cell with an effective amount of a splicingmodulator, a splicing modulator-based antibody-drug conjugate (ADC), ora composition comprising a splicing modulator or ADC. In variousembodiments, the present disclosure provides a method of inducingimmunogenic cell death by contacting a neoplastic cell with an effectiveamount of a splicing modulator, a splicing modulator-based antibody-drugconjugate (ADC), or a composition comprising a splicing modulator orADC.

In some embodiments, the at least one neoantigen comprises an amino acidsequence of any one of SEQ ID NOs: 37-65. In some embodiments, the atleast one neoantigen comprises an amino acid sequence of SEQ ID NO: 37.In some embodiments, the at least one neoantigen comprises an amino acidsequence of SEQ ID NO: 39. In some embodiments, the at least oneneoantigen comprises an amino acid sequence of any one of SEQ ID NOs:46-49.

In some embodiments, the neoplastic cell is present in an in vitro cellculture. In some embodiments, the neoplastic cell is obtained from asubject. In some embodiments, the neoplastic cell is present in asubject. In some embodiments, the neoplastic cell is derived from ahematological malignancy or a solid tumor. In some embodiments, thehematological malignancy is selected from a B-cell malignancy, aleukemia, a lymphoma, and a myeloma. In some embodiments, thehematological malignancy is selected from acute myeloid leukemia andmultiple myeloma. In some embodiments, the solid tumor is selected frombreast cancer (e.g., HER2-positive breast cancer), gastric cancer (e.g.,gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer(e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serousendometrial carcinoma), salivary duct carcinoma, melanoma, colon cancer,cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer,and esophageal cancer. In some embodiments, the solid tumor is selectedfrom HER2-positive breast cancer, gastric adenocarcinoma, prostatecancer, and osteosarcoma.

In various embodiments, the present disclosure further provides a methodof inducing at least one neoantigen and/or a T-cell response in asubject having or suspected of having a neoplastic disorder byadministering to the subject an effective amount of a splicingmodulator, an ADC, or a composition comprising a splicing modulator orADC. Also provided herein, in various embodiments, is a method oftreating a subject having or suspected of having a neoplastic disorderby administering to the subject an effective amount of a splicingmodulator, an ADC, or a composition comprising a splicing modulator orADC, wherein administration of the splicing modulator, antibody-drugconjugate, or composition induces at least one neoantigen and/or aT-cell response.

In various other embodiments, the present disclosure provides a methodof inducing a double-stranded RNA immune response in a subject having orsuspected of having a neoplastic disorder by administering to thesubject an effective amount of a splicing modulator, an ADC, or acomposition comprising a splicing modulator or ADC. Also providedherein, in various embodiments, is a method of treating a subject havingor suspected of having a neoplastic disorder by administering to thesubject an effective amount of a splicing modulator, an ADC, or acomposition comprising a splicing modulator or ADC, whereinadministration of the splicing modulator, antibody-drug conjugate, orcomposition induces a double-stranded RNA immune response.

In still other embodiments, the present disclosure provides a method ofinducing immunogenic cell death in a subject having or suspected ofhaving a neoplastic disorder by administering to the subject aneffective amount of a splicing modulator, an ADC, or a compositioncomprising a splicing modulator or ADC. Further provided herein, invarious embodiments, is a method of treating a subject having orsuspected of having a neoplastic disorder by administering to thesubject an effective amount of a splicing modulator, an ADC, or acomposition comprising a splicing modulator or ADC, whereinadministration of the splicing modulator, antibody-drug conjugate, orcomposition induces immunogenic cell death.

In some embodiments of the therapeutic methods described herein, the atleast one neoantigen comprises an amino acid sequence of any one of SEQID NOs: 37-65. In some embodiments, the at least one neoantigencomprises an amino acid sequence of SEQ ID NO: 37. In some embodiments,the at least one neoantigen comprises an amino acid sequence of SEQ IDNO: 39. In some embodiments, the at least one neoantigen comprises anamino acid sequence of any one of SEQ ID NOs: 46-49.

In some embodiments, the present disclosure further provides a method oftreating a subject having or suspected of having a neoplastic disorderby administering to the subject an effective amount of a splicingmodulator, an ADC, or a composition comprising a splicing modulator orADC, wherein administration of the splicing modulator, antibody-drugconjugate, or composition induces immunogenic cell death, in combinationwith one or more additional therapies comprising a second agent.

In some embodiments of the therapeutic methods described herein, theamount of the splicing modulator, antibody-drug conjugate, composition,or second agent administered is reduced due to induction of at least oneneoantigen and/or a T-cell response, as compared to a standard dosage ofthe splicing modulator, antibody-drug conjugate, composition, or secondagent. In some embodiments, the administered amount of the splicingmodulator, antibody-drug conjugate, composition, or second agent isreduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, ascompared to a standard dosage of the splicing modulator, antibody-drugconjugate, composition, or second agent. In some embodiments, thesplicing modulator, antibody-drug conjugate, composition, or secondagent is administered at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 75%, or 90% less frequently, as compared to a standard dosingregimen of the splicing modulator, antibody-drug conjugate, composition,or second agent. In some embodiments, the administered amount and/ordosage of the splicing modulator, antibody-drug conjugate, composition,or second agent results in lower systemic toxicity and/or improvedtolerance.

As used herein, the term “standard dosage” or “standard dosing regimen”refers to any usual or routine dosing regimen for a therapeutic agent,e.g., a regimen proposed by the manufacturer, approved by regulatoryauthorities, or otherwise tested in human subjects to meet the averagepatient's needs. In some embodiments, the therapeutic agent is asplicing modulator, an antibody, or an antibody-drug conjugate withanti-cancer activity.

For instance, a standard dosing regimen for trastuzumab, an exemplaryanti-HER2 antibody disclosed herein, may be 8 mg/kg administeredintravenously over 90 min (week 1) followed by 6 mg/kg administeredintravenously over 30-90 min every 3 weeks (week 4 through the end ofthe therapy cycle) (Herceptin® (trastuzumab) FDA Label Supplement,2017).

As another example, a standard dosing regimen for ipilimumab, anexemplary anti-CTLA4 checkpoint inhibitor antibody, may be 3 mg/kgadministered intravenously over 90 min every 3 weeks for 4 doses(Yervoy® (ipilimumab) FDA Label Supplement, 2018). Another standarddosing regimen for ipilimumab may be 10 mg/kg administered intravenouslyover 90 min every 3 weeks for 4 doses, followed by 10 mg/kg every 12weeks for up to 3 years (Yervoy® (ipilimumab) FDA Label Supplement,2018).

As another example, a standard dosing regimen for nivolumab, anexemplary anti-PD1 checkpoint inhibitor antibody, may be 3 mg/kgadministered intravenously over 60 min every 2 weeks (Opdivo®(nivolumab) FDA Label, 2015).

As another example, a standard dosing regimen for atezolizumab, anexemplary anti-PDL1 checkpoint inhibitor antibody, may be 1200 mgadministered intravenously over 60 min every 3 weeks (Tecentriq®(atezolizumab) FDA Label Supplement, 2018).

As yet another example, a standard dosing regimen for T-DM1, anexemplary anti-HER2 antibody-drug conjugate, may be 3.6 mg/kgadministered intravenously over 90 min every 3 weeks (Kadcyla® (T-DM1)FDA Label Supplement, 2016).

In some embodiments, the methods described herein may further compriseadministering at least one additional therapy (e.g., a checkpointinhibitor, a neoantigen vaccine, a cytokine or cytokine analog, CAR-T,etc.). In some embodiments, the amount of the splicing modulator,antibody-drug conjugate, composition, and/or the at least one additionaltherapy administered is reduced due to induction of at least oneneoantigen and/or a T-cell response, as compared to a standard dosage ofthe splicing modulator, antibody-drug conjugate, composition, and/or theat least one additional therapy. In some embodiments, the amount of thesplicing modulator, antibody-drug conjugate, composition, and/or the atleast one additional therapy administered is reduced due to induction ofa double-stranded RNA immune response, as compared to a standard dosageof the splicing modulator, antibody-drug conjugate, composition, and/orthe at least one additional therapy. In some embodiments, the amount ofthe splicing modulator, antibody-drug conjugate, composition, and/or theat least one additional therapy administered is reduced due to inductionof immunogenic cell death, as compared to a standard dosage of thesplicing modulator, antibody-drug conjugate, composition, and/or the atleast one additional therapy. In some embodiments, the administeredamount of the splicing modulator, antibody-drug conjugate, composition,and/or the at least one additional therapy is reduced by 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, as compared to a standarddosage of the splicing modulator, antibody-drug conjugate, composition,and/or the at least one additional therapy. In some embodiments, thesplicing modulator, antibody-drug conjugate, composition, and/or the atleast one additional therapy is administered at least 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% less frequently, as comparedto a standard dosing regimen of the splicing modulator, antibody-drugconjugate, composition, and/or the at least one additional therapy. Insome embodiments, the administered amount and/or dosage of the splicingmodulator, antibody-drug conjugate, composition, and/or the at least oneadditional therapy results in lower systemic toxicity and/or improvedtolerance.

In some embodiments, administration of the splicing modulator,antibody-drug conjugate, or composition is initiated beforeadministration of the at least one additional therapy. In otherembodiments, administration of the splicing modulator, antibody-drugconjugate, or composition is initiated after administration of the atleast one additional therapy. In still other embodiments, administrationof the splicing modulator, antibody-drug conjugate, or composition isinitiated concurrently with administration of the at least oneadditional therapy.

In some embodiments, administration of the splicing modulator,antibody-drug conjugate, or composition is repeated at least once afterinitial administration. In some embodiments, the amount of the splicingmodulator, antibody-drug conjugate, or composition used for repeatedadministration is reduced as compared to the amount used for initialadministration. In some embodiments, the amount of the splicingmodulator, antibody-drug conjugate, or composition used for repeatedadministration is reduced as compared to a standard dosage of thesplicing modulator, antibody-drug conjugate, or composition. In someembodiments, the amount of the splicing modulator, antibody-drugconjugate, or composition used for repeated administration is reduced by10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, as compared toa standard dosage or initial dosage of the splicing modulator,antibody-drug conjugate, or composition.

In some embodiments, administration of the at least one additionaltherapy is repeated at least once after initial administration. In someembodiments, the amount of the at least one additional therapy used forrepeated administration is reduced as compared to the amount used forinitial administration. In some embodiments, the amount of the at leastone additional therapy used for repeated administration is reduced ascompared to a standard dosage of the at least one additional therapy. Insome embodiments, the amount of the at least one additional therapy usedfor repeated administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 75%, or 90%, as compared to a standard dosage or initialdosage of the at least one additional therapy.

In some embodiments, repeated administration of the splicing modulator,antibody-drug conjugate, or composition is concurrent with repeatedadministration of the at least one additional therapy. In someembodiments, administration of the splicing modulator, antibody-drugconjugate, or composition is sequential or staggered with repeatedadministration of the at least one additional therapy.

In some embodiments, the at least one additional therapy comprisesadministering a checkpoint inhibitor, e.g., any checkpoint inhibitordisclosed herein. In some embodiments, the subject is intolerant,non-responsive, or poorly responsive to the checkpoint inhibitor whenadministered alone. In some embodiments, the checkpoint inhibitor istargeted at PD1/PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and/or KIR.In some embodiments, the checkpoint inhibitor is targeted at CTLA4,OX40, CD40, and/or GITR. In some embodiments, the checkpoint inhibitoris an antibody having inhibitory or agonist activity to its target. Insome embodiments, a checkpoint inhibitor is targeted with an inhibitoryantibody or other similar inhibitory molecule. In other embodiments, acheckpoint inhibitor is targeted with an agonist antibody or othersimilar agonist molecule.

In some other embodiments, the at least one additional therapy comprisesadministering a neoantigen vaccine, e.g., any neoantigen vaccinedisclosed herein. In some embodiments, the splicing modulator, ADC, orcomposition is administered before administration of the neoantigenvaccine. In some embodiments, the splicing modulator, ADC, orcomposition is administered after administration of the neoantigenvaccine. In some embodiments, the splicing modulator, ADC, orcomposition is administered concurrently with administration of theneoantigen vaccine. In some embodiments, administration of the splicingmodulator, ADC, or composition is repeated at least once after initialadministration. In some embodiments, the amount of the splicingmodulator, ADC, or composition used for repeated administration isreduced as compared to the amount used for initial administration.

In some embodiments, the neoantigen vaccine comprises at least oneneoantigen peptide. In some embodiments, the at least one neoantigenpeptide ranges from about 10 to about 50 amino acids in length. In someembodiments, the at least one neoantigen peptide ranges from about 10 toabout 35 amino acids in length. In some embodiments, the at least oneneoantigen peptide ranges from about 15 to about 25 amino acids inlength. In some embodiments, the at least one neoantigen peptidecomprises one or more than one neoantigen sequence.

In some embodiments, the neoantigen sequence comprises an amino acidsequence of any one of SEQ ID NOs: 37-65. In some embodiments, theneoantigen sequence comprises an amino acid sequence of SEQ ID NO: 37.In some embodiments, the neoantigen sequence comprises an amino acidsequence of SEQ ID NO: 39. In some embodiments, the neoantigen sequencecomprises an amino acid sequence of any one of SEQ ID NOs: 46-49.

In some other embodiments, the neoantigen sequence comprises an aminoacid sequence of any one of SEQ ID NOs: 66-93, or an antigenic portionof any one of SEQ ID NOs: 66-93. In some embodiments, the neoantigensequence comprises an amino acid sequence of SEQ ID NO: 66, or anantigenic portion of SEQ ID NO: 66. In some embodiments, the neoantigensequence comprises an amino acid sequence of any one of SEQ ID NOs:74-77, or an antigenic portion of any one of SEQ ID NOs: 74-77. In someembodiments, the neoantigen sequence and/or antigenic portion rangesfrom about 10 to about 50 amino acids in length. In some embodiments,the neoantigen sequence and/or antigenic portion ranges from about 10 toabout 35 amino acids in length. In some embodiments, the neoantigensequence and/or antigenic portion ranges from about 15 to about 25 aminoacids in length. In some embodiments, the neoantigen sequence and/orantigenic portion ranges from about 10 to about 20 amino acids inlength. In some embodiments, the neoantigen sequence and/or antigenicportion does not exclusively overlap or consist of the canonical peptidesequence (e.g., any of the exemplary canonical peptide sequencesunderlined in Table 21).

The term “antigenic portion” or “antigenic fragment” of a neoantigensequence, as used herein, refers to one or more fragments of aneoantigen sequence that retain the ability to induce a T-cell response(e.g., antigen-specific expansion and/or maturation of effector T-cellpopulation(s)). An antigenic portion, in some embodiments, may alsoretain the ability to be internalized, processed, and/or presented byantigen-presenting cells (e.g., dendritic cells). In some embodiments,an antigenic portion also retains T-cell priming function. In someembodiments, an antigenic portion of a neoantigen sequence ranges fromabout 10 to about 50 amino acids in length. In some embodiments, anantigenic portion of a neoantigen sequence ranges from about 10 to about35 amino acids in length. In some embodiments, an antigenic portion of aneoantigen sequence ranges from about 15 to about 25 amino acids inlength. In some embodiments, an antigenic portion of a neoantigensequence ranges from about 10 to about 20 amino acids in length. In someembodiments, an antigenic portion of a neoantigen sequence (e.g., anantigenic portion of any one of SEQ ID NOs: 66-93), or its encodingmRNA, is formulated as a neoantigen vaccine.

An exemplary embodiment of an antigenic portion is the region(s)flanking amino acids 45-53 of SEQ ID NO: 66. Another exemplaryembodiment of an antigenic portion is the region(s) flanking amino acids82-90 of SEQ ID NO: 66. In some embodiments, the antigenic portion iscapable of binding to at least one HLA allele expressed in a subject(e.g., HLA-A*02:01). In some other embodiments, the antigenic portion iscapable of binding to at least one HLA allele expressed in at least 10%,at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, or at least 45% of subjects in a population of subjectssuffering from a neoplastic disorder. In some embodiments, the antigenicportion is capable of eliciting a T-cell response against a tumorpresent in at least 1%, at least 5%, or at least 10% of a population ofsubjects suffering from a neoplastic disorder.

In some embodiments, an antigenic portion does not exclusively overlapor consist of a canonical peptide sequence. The term “canonical peptidesequence,” as used herein, refers to any contiguous peptide sequencepresent in the human proteome in the absence of contact with a splicingmodulator (e.g., in the absence of contact with a splicing modulatoralone and/or as part of an ADC or composition), and/or to which theimmune has previously been exposed. In some embodiments, the canonicalpeptide sequence is derived from and/or encoded by the canonicaltranscript open reading frame. Exemplary canonical peptide sequences areunderlined in Table 21.

In some embodiments, when a splicing modulator is administered (e.g.,alone and/or as part of an ADC or composition), a canonical peptidesequence may be derived from and/or encoded by the immediate 5′ in-frame24 nucleotides preceding an aberrant splicing event induced by thesplicing modulator. Thus, in some embodiments, the canonical peptidesequence comprises or consists of the 8 amino acids immediatelyN-terminal to the neoantigen sequence induced by the splicing modulator.In some embodiments, when a 5′ exon sequence terminates with a terminalnucleotide of a codon, the canonical peptide sequence terminates at theend of the exon. In some other embodiments, when a 5′ exon sequenceterminates with one or two of the three nucleotides of a codon, thecanonical peptide sequence is derived from and/or encoded by the 24nucleotides preceding the incomplete codon. In some embodiments, mRNAsequences 3′ of the aberrant splicing event may be translated in thesame open reading frame derived from the 5′ exon until reaching a stopcodon, whereupon translation may terminate. In some embodiments, whenthe aberrant splicing event (e.g., exon skipping) results in aconservation of the canonical transcript open reading frame, theC-terminal sequence may be translated for an additional 24 nucleotides,encoding 8 C-terminal amino acids. In this context, in some embodiments,only the region across the aberrant exon junction may encode aneoantigen sequence. In some embodiments, when the open reading frame isshifted (e.g., intron retention), the complete C-terminal sequence(encoded by the 3′ mRNA) may encode a neoantigen sequence.

In some embodiments, an antigenic portion of a neoantigen sequence ischosen by comparing the neoantigen sequence to the canonical peptidesequence; and selecting a portion of the neoantigen sequence that doesnot exclusively overlap, consist of, and/or align with the canonicalpeptide sequence. An antigenic portion of a neoantigen sequence, in someembodiments, can be screened for antigenicity and/or T-cell primingfunction in the same manner as are full-length neoantigen sequences(e.g., the neoantigen sequence from which the antigenic portion isderived). In some embodiments, an antigenic portion of a neoantigensequence is evaluated for antigenicity and/or T-cell priming functionusing a T-cell priming assay, such as the exemplary T-cell primingexperiments described herein.

In some embodiments, the neoantigen sequence is a neoantigen sequencespecific to the subject. In some embodiments, the neoantigen sequence isa personalized neoantigen vaccine for the subject. In some embodiments,the neoantigen sequence used to create a personalized neoantigen vaccinefor a subject is capable of binding to at least one HLA allele expressedin the subject. In some embodiments, a personalized neoantigen vaccineis selected by identifying neoantigens expressed in a subject's tumor,e.g., after administration of a splicing modulator or ADC, and selectinga vaccine comprising a neoantigen sequence observed in the patient'stumor, e.g., a vaccine comprising an amino acid sequence of any one ofSEQ ID NOs: 37-65 that is observed in the tumor.

The term “personalized” when used to describe a neoantigen vaccinerefers to a vaccine created by identifying one or more neoantigensproduced in a patient, preferably one identified in the patient after anexposure to a splicing modulator, ADC, or composition, and then usingone or more of those neoantigens as the basis of the vaccine for thesame patient. Accordingly, in some embodiments, a patient is given asplicing modulator, ADC, or composition and screened for neoantigensproduced by the treatment. In some embodiments, the selected neoantigenvaccine comprises a neoantigen peptide or mRNA disclosed herein andconfirmed to be present in the patient after exposure to the splicingmodulator, ADC, or composition. In some embodiments, the splicingmodulator, ADC, or composition and/or peptide or mRNA vaccine may beadministered to the patient once or repeatedly. Subsequently, in someembodiments, one or more of those neoantigens are used to create apersonalized vaccine that is given to the patient. In some embodiments,the one or more neoantigens used to create a personalized vaccinepossess binding affinity for one or more patient-specific HLA alleles.In some embodiments, the patient expresses one or more MHC1 alleles thatbind to the one or more neoantigens. The prediction of whether a givenneoantigen will bind to a specific MHC1 allele can be determined usingany computational prediction method known in the art. Exemplarycomputational prediction methods are disclosed, e.g., in Meydan et al.(2013) BMC Bioinformatics 14(Suppl. 2):513, which is incorporated hereinby reference for such methods.

In some other embodiments, the neoantigen sequence is a universalneoantigen sequence. In some embodiments, the neoantigen sequence is auniversal neoantigen vaccine.

The term “universal” when used to describe a neoantigen vaccine refersto a vaccine having a peptide or mRNA sequence that is based on commonor known neoantigen(s) observed by sequencing neoantigens produced inmultiple patients and/or patient tissue samples, preferably after anexposure to a splicing modulator, ADC, or composition. The peptide ormRNA sequence used in the vaccine need not be present in every patientbut rather be observed in at least several patients or patient tissuesamples. In some embodiments, the splicing modulator, ADC, orcomposition and/or peptide or mRNA vaccine may be administered to thepatient once or repeatedly. Subsequently, in some embodiments, thatpeptide or mRNA sequence is used for vaccinating further patients. Insome embodiments, a patient is given a splicing modulator, ADC, orcomposition, and then given a peptide or mRNA vaccine of knownneoantigen to enhance immune response to the neoantigens produced by thesplicing modulator, ADC, or composition. In some embodiments, a patientis given a universal peptide or mRNA vaccine and then given a splicingmodulator, ADC, or composition once or repeatedly. In some embodiments,the neoantigen sequence (or sequences) used to create a universalneoantigen vaccine is selected based on overall MHC1 allele frequency ina given patient population (Maiers et al. (2007) Hum. Immunol.68(9):779-88).

In some embodiments, the neoantigen (e.g., a universal neoantigen)sequence is capable of binding to at least one HLA allele expressed inat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% of subjects in a population ofsubjects suffering from the neoplastic disorder. In some embodiments,the neoantigen sequence is capable of eliciting a T-cell responseagainst a tumor present in at least 1%, at least 5%, or at least 10% ofa population of subjects suffering from the neoplastic disorder.

In some embodiments, the neoantigen sequence has been identified bysequencing at least one neoantigen peptide, or its encoding mRNA,induced in the subject by administering an effective amount of thesplicing modulator, antibody-drug conjugate, or composition. In someembodiments, the at least one neoantigen peptide comprises a neoantigensequence induced by contacting a neoplastic cell with an effectiveamount of the splicing modulator, antibody-drug conjugate, orcomposition. In some embodiments, the neoplastic cell is present in anin vitro cell culture. In some embodiments, the neoplastic cell isobtained from the subject. In some embodiments, the neoplastic cell ispresent in the subject.

In some embodiments, the neoantigen vaccine comprises at least oneneoantigen peptide and a pharmaceutically acceptable carrier (e.g., anyof the exemplary carriers described herein). In some embodiments, the atleast one neoantigen peptide is linked to the pharmaceuticallyacceptable carrier. In some embodiments, the pharmaceutically acceptablecarrier is selected from a peptide, a serum albumin, a keyhole limpethemocyanin, an immunoglobulin, a thyroglobulin, an ovalbumin, a toxoidor an attenuated toxoid derivative, a cytokine, and a chemokine. In someembodiments, the neoantigen peptide and the pharmaceutically acceptablecarrier are covalently attached via a linker. In some embodiments, theneoantigen peptide and the pharmaceutically acceptable carrier areexpressed as a fusion protein. In some embodiments, the neoantigenvaccine comprises at least one neoantigen peptide and a pharmaceuticallyacceptable diluent. In some embodiments, the neoantigen vaccinecomprises at least one neoantigen peptide and a pharmaceuticallyacceptable adjuvant.

In some embodiments, the neoantigen vaccine comprises at least oneneoantigen mRNA. In some embodiments, the at least one neoantigen mRNAencodes one or more than one neoantigen sequence. In some embodiments,the neoantigen sequence comprises an amino acid sequence of any one ofSEQ ID NOs: 37-65. In some embodiments, the neoantigen sequencecomprises an amino acid sequence of SEQ ID NO: 37. In some embodiments,the neoantigen sequence comprises an amino acid sequence of SEQ ID NO:39. In some embodiments, the neoantigen sequence comprises an amino acidsequence of any one of SEQ ID NOs: 46-49.

In some other embodiments, the neoantigen sequence comprises an aminoacid sequence of any one of SEQ ID NOs: 66-93, or an antigenic portionof any one of SEQ ID NOs: 66-93. In some embodiments, the neoantigensequence comprises an amino acid sequence of SEQ ID NO: 66, or anantigenic portion of SEQ ID NO: 66. In some embodiments, the neoantigensequence comprises an amino acid sequence of any one of SEQ ID NOs:74-77, or an antigenic portion of any one of SEQ ID NOs: 74-77. In someembodiments, the neoantigen sequence and/or antigenic portion rangesfrom about 10 to about 50 amino acids in length. In some embodiments,the neoantigen sequence and/or antigenic portion ranges from about 10 toabout 35 amino acids in length. In some embodiments, the neoantigensequence and/or antigenic portion ranges from about 15 to about 25 aminoacids in length. In some embodiments, the neoantigen sequence and/orantigenic portion ranges from about 10 to about 20 amino acids inlength. In some embodiments, the neoantigen sequence and/or antigenicportion does not exclusively overlap or consist of the canonical peptidesequence (e.g., any of the exemplary canonical peptide sequencesunderlined in Table 21).

In some embodiments, the neoantigen sequence is a neoantigen sequencespecific to the subject. In some embodiments, the neoantigen sequence isa personalized neoantigen vaccine for the subject. In some embodiments,the neoantigen sequence is capable of binding to at least one HLA alleleexpressed in the subject.

In some other embodiments, the neoantigen sequence is a universalneoantigen sequence. In some embodiments, the neoantigen sequence is auniversal neoantigen vaccine. In some embodiments, the neoantigensequence is capable of binding to at least one HLA allele expressed inat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% of subjects in a population ofsubjects suffering from the neoplastic disorder. In some embodiments,the neoantigen sequence is capable of eliciting a T-cell responseagainst a tumor present in at least 1%, at least 5%, or at least 10% ofa population of subjects suffering from the neoplastic disorder.

In some embodiments, the neoantigen sequence has been identified bysequencing the protein sequence of at least one neoantigen. In someembodiments, the neoantigen sequence has been identified by sequencingat least one mRNA encoding a neoantigen induced in the subject byadministering an effective amount of the splicing modulator,antibody-drug conjugate, or composition. In some embodiments, the atleast one neoantigen mRNA encodes a neoantigen sequence induced bycontacting a neoplastic cell with an effective amount of the splicingmodulator, antibody-drug conjugate, or composition. In some embodiments,the neoplastic cell is present in an in vitro cell culture. In someembodiments, the neoplastic cell is obtained from the subject. In someembodiments, the neoplastic cell is present in the subject.

In some embodiments, the neoantigen vaccine comprises at least oneneoantigen mRNA and a pharmaceutically acceptable carrier (e.g., any ofthe exemplary carriers described herein). In some embodiments, the atleast one neoantigen mRNA is linked to the pharmaceutically acceptablecarrier. In some embodiments, the pharmaceutically acceptable carrier isselected from a peptide, a serum albumin, a keyhole limpet hemocyanin,an immunoglobulin, a thyroglobulin, an ovalbumin, a toxoid or anattenuated toxoid derivative, a cytokine, and a chemokine. In someembodiments, the neoantigen vaccine comprises at least one neoantigenmRNA and a pharmaceutically acceptable diluent. In some embodiments, theneoantigen vaccine comprises at least one neoantigen mRNA and apharmaceutically acceptable adjuvant. In some embodiments, theneoantigen mRNA is encapsulated by an encapsulating agent. In someembodiments, the encapsulating agent is a liposome. In some embodiments,the encapsulating agent is a nanoparticle.

In some embodiments, the at least one additional therapy comprisesadministering a cytokine or cytokine analog, e.g., any cytokine orcytokine analog disclosed herein. In some embodiments, the subject isintolerant, non-responsive, or poorly responsive to the cytokine orcytokine analog when administered alone. In some embodiments, thecytokine or cytokine analog comprises a T-cell enhancer. In someembodiments, the cytokine or cytokine analog comprises IL-2, IL-10,IL-12, IL-15, IFNγ, and/or TNFα. In some embodiments, the cytokine orcytokine analog comprises IL-2, IL-10, IL-12, and/or IL-15. In someembodiments, administering the cytokine or cytokine analog enhancesT-cell priming following administration of a splicing modulator,antibody-drug conjugate, or composition due to the induction andpresentation of neoantigens.

In some embodiments, the at least one additional therapy comprisesadministering engineered tumor-targeting T-cells (i.e., CAR-T), e.g.,any CAR-T therapy disclosed herein.

In some embodiments, the methods described herein may further comprisedetecting one or more neoantigens and/or a T-cell response in thesubject after administration of the splicing modulator, antibody-drugconjugate, or composition, and, optionally, continuing administration ofthe splicing modulator, antibody-drug conjugate, or composition if oneor more neoantigens and/or a T-cell response is detected. In someembodiments, detecting one or more neoantigens and/or a T-cell responsein the subject indicates efficacy of treatment with the splicingmodulator, antibody-drug conjugate, or composition. In some embodiments,treatment with the additional therapy, along with splicing modulator,antibody-drug conjugate, or composition, is continued if one or moreneoantigens and/or a T-cell response is detected. In some embodiments,treatment is continued at a reduced dosage and/or frequency if one ormore neoantigens and/or a T-cell response is detected.

In some embodiments, the methods described herein may further comprisedetecting a double-stranded RNA immune response in the subject afteradministration of the splicing modulator, antibody-drug conjugate, orcomposition, and, optionally, continuing administration of the splicingmodulator, antibody-drug conjugate, or composition if a double-strandedRNA immune response is detected. In some embodiments, detecting adouble-stranded RNA immune response in the subject indicates efficacy oftreatment with the splicing modulator, antibody-drug conjugate, orcomposition. In some embodiments, treatment with the additional therapy,along with splicing modulator, antibody-drug conjugate, or composition,is continued if a double-stranded RNA immune response is detected. Insome embodiments, treatment is continued at a reduced dosage and/orfrequency if a double-stranded RNA immune response is detected.

In some embodiments, the methods described herein may further comprisedetecting immunogenic cell death in the subject after administration ofthe splicing modulator, antibody-drug conjugate, or composition, and,optionally, continuing administration of the splicing modulator,antibody-drug conjugate, or composition if immunogenic cell death isdetected. In some embodiments, detecting immunogenic cell death in thesubject indicates efficacy of treatment with the splicing modulator,antibody-drug conjugate, or composition. In some embodiments, treatmentwith the additional therapy, along with splicing modulator,antibody-drug conjugate, or composition, is continued if immunogeniccell death is detected. In some embodiments, treatment is continued at areduced dosage and/or frequency if immunogenic cell death is detected.

In some embodiments, the subject has a non-synonymous mutational burdenof about 150 mutations or less. In some embodiments, the subject has anon-synonymous mutational burden of about 100 mutations or less. In someembodiments, the subject has a non-synonymous mutational burden of about50 mutations or less. In some embodiments, the subject has or issuspected of having a neoplastic disorder, e.g., a hematologicalmalignancy or a solid tumor. In some embodiments, the hematologicalmalignancy is selected from a B-cell malignancy, a leukemia, a lymphoma,and a myeloma. In some embodiments, the hematological malignancy isselected from acute myeloid leukemia and multiple myeloma. In someembodiments, the solid tumor is selected from breast cancer, gastriccancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer,salivary duct carcinoma, melanoma, colon cancer, cervical cancer,pancreatic cancer, kidney cancer, colorectal cancer, and esophagealcancer. In some embodiments, the solid tumor is selected fromHER2-positive breast cancer, gastric adenocarcinoma, prostate cancer,and osteosarcoma.

In various embodiments, the present disclosure further provides a methodof treating a subject having or suspected of having a neoplasticdisorder, comprising: (a) administering to the subject an effectiveamount of a splicing modulator, an ADC, or composition comprising asplicing modulator or ADC, wherein administration of the splicingmodulator, antibody-drug conjugate, or composition induces at least oneneoantigen and/or a T-cell response; (b) detecting one or moreneoantigens and/or a T-cell response in the subject after administrationof the splicing modulator, antibody-drug conjugate, or composition; and(c) continuing administration of the splicing modulator, antibody-drugconjugate, or composition if one or more neoantigens and/or a T-cellresponse is detected. In some embodiments, detecting one or moreneoantigens and/or a T-cell response in the subject indicates efficacyof treatment with the splicing modulator, antibody-drug conjugate, orcomposition. In some embodiments, the one or more neoantigens comprisean amino acid sequence of any one of SEQ ID NOs: 37-65. In someembodiments, the one or more neoantigens comprise an amino acid sequenceof SEQ ID NO: 37. In some embodiments, the one or more neoantigenscomprise an amino acid sequence of SEQ ID NO: 39. In some embodiments,the one or more neoantigens comprise an amino acid sequence of any oneof SEQ ID NOs: 46-49.

Combination of Splicing Modulator/ADC and Immune Checkpoint Inhibition:

In various embodiments, a patient having a cancer as described hereincan be treated with a combination of a splicing modulator, ADC, orcomposition and a checkpoint inhibitor therapy. Immune checkpoints areinhibitory pathways that slow down or stop immune reactions and preventexcessive tissue damage from uncontrolled activity of immune cells. Asused herein, the term “checkpoint inhibitor” is meant to refer to anytherapeutic agent, including any small molecule chemical compound,antibody, nucleic acid molecule, or polypeptide, or any fragmentsthereof, that inhibits one or more of the inhibitory pathways, therebyallowing more extensive immune activity.

Treatment of patients with immune checkpoint inhibition has been shownto have robust efficacy in certain clinical indications. Recently, theFDA approved use of a checkpoint inhibitor in patients with tumorsexhibiting high microsatellite instability, agnostic to the tissuelineage. This approval was based, in part, on the observation thatresponse rates correlate positively with mutational burden (Rizvi et al.(2015) Science 348(6230):124-8; Hellmann et al. (2018) Cancer Cell33(5):853-861). Estimates from the literature vary in absolute numbersand by lineage, but generally support that above a threshold of ˜150-250mutations, the probability of response rises. Analysis of TOGA datashows that a large percentage of adult-onset tumor lineages havecomparatively low non-synonymous mutational burden (Vogelstein et al.(2013) Science 339:1549-58). Most lineages have median non-synonymousmutational rates of ˜30-80 per patient, well below the thresholds forimproved odds of response to checkpoint inhibitors.

For instance, HER2-positive breast cancer has been shown to have amedian of ˜60 non-synonymous mutations present per patient sample.However, the threshold for checkpoint inhibitor treatment efficacy, asmentioned above, is estimated to be in the range of ˜150-250non-synonymous mutations, i.e., patients above this threshold are morelikely to show complete remission, partial remission, and/or stabledisease, whereas patients below this threshold are more likely toexhibit progressive disease. Strategies to enhance the apparent numberof non-synonymous mutations and/or neoantigens being presented on tumorcells are therefore desirable, and may enhance the overall probabilityof response, e.g., to checkpoint inhibitor therapies. As cytokines (andanalogs thereof) act via a similar mechanism of action, such strategiesmay also enhance the overall probability of response to cytokine-basedtherapies.

Current response rates in HER2-positive breast cancer are ˜15-25% (CTINCT02129556). In various embodiments disclosed herein, treatment with asplicing modulator, ADC, or composition in combination with a checkpointinhibitor and/or cytokine therapy may improve such response rates. Invarious embodiments, treatment with a splicing modulator, ADC, orcomposition in combination with a checkpoint inhibitor and/or cytokinetherapy may apply to any adult-onset tumor, particularly those in whichthe median non-synonymous mutational rate is below the estimated ˜150mutations threshold. In various embodiments, exemplary cancer typessuitable for treatment with a splicing modulator, ADC, or composition ofthe present disclosure, alone or in combination with an additionaltherapy (e.g., a checkpoint inhibitor therapy, a cytokine therapy)include but are not limited to esophageal cancer, non-Hodgkin'slymphoma, colorectal cancer, head and neck cancer, gastric cancer,endometrial cancer, pancreatic adenocarcinoma, ovarian cancer, prostatecancer, hepatocellular cancer, glioblastoma, breast cancer (e.g.,HER2-positive breast cancer), lung cancer (e.g., non-small cell lungcancer), chronic lymphocytic leukemia, and acute myeloid leukemia. Otherexemplary suitable cancer types are identified, e.g., in Vogelstein etal. (2013) Science 339:1549-58, which is incorporated herein byreference in its entirety.

As many checkpoint inhibitor therapies are based on chronic expressionof tumor-associated antigens, regular treatment boosts are required forefficacy and for “re-boosting” reactive T-cell populations. Theinducible nature of splicing modulator or ADC-derived neoantigensdescribed herein provide for therapeutic dosing regimens that may bedesigned to enhance the immune response of neoantigen-reactive T-cells,while limiting T-cell exhaustion often caused by chronic antigenstimulation. For instance, in some embodiments, an initial dose of asplicing modulator, ADC, or composition is administered to a subject totrigger aberrant splicing and production of neoantigen peptides. After aperiod of time to allow for protein production and antigen presentation,in some embodiments, the subject is then administered an initial dose ofa checkpoint inhibitor to boost and/or enhance effector T-cell primingand expansion. In some embodiments, the wait period between doses ofsplicing modulator, ADC, or composition and checkpoint inhibitor isabout 2, about 3, about 4, about 5, about 6, or about 7 days. In someembodiments, the wait period is between about 3 days and about 5 days.In some embodiments, the checkpoint inhibitor is targeted at CTLA4,OX40, CD40, and/or GITR. In some embodiments, the combinationtherapeutic benefit of a splicing modulator, ADC, or composition and acheckpoint inhibitor may be additive or superadditive.

In some embodiments, after a period to allow for T-cell priming andexpansion, the subject is then administered a second or subsequent doseof the splicing modulator, ADC, or composition to triggerre-presentation of neoantigen peptides. In some embodiments, the waitperiod between an initial dose of a checkpoint inhibitor and a second orsubsequent dose of a splicing modulator, ADC, or composition is about 2,about 3, about 4, or about 5 weeks. In some embodiments, the wait periodis about 3 weeks. Following a second or subsequent dose of the splicingmodulator, ADC, or composition, in some embodiments, the immune systemmay engage with the neoantigen-presenting tumor cells and/or elicittumor cell killing. In some embodiments, the subject is thenadministered a second or subsequent dose of the checkpoint inhibitor tofurther expand the memory effector T-cell population, after allowing forsecondary T-cell priming and expansion.

In some embodiments, dosing of the splicing modulator, ADC, orcomposition following this exemplary initial treatment regimen can bepulsatile, i.e., the splicing modulator, ADC, or composition may bedosed at prolonged intervals (e.g., about every 4 weeks, about every 5weeks, about every 6 weeks) to allow for antigen presentation, T-cellengagement and/or tumor cell killing, and/or recovery of the memoryT-cell population. At later timepoints, in some embodiments, thesplicing modulator, ADC, or composition treatment may be combined withone or more checkpoint inhibitors targeted to restore effectorfunctionality to exhausted T-cell populations. For example, in someembodiments, at later timepoints, the splicing modulator, ADC, orcomposition treatment may be combined with one or more checkpointinhibitors targeted at PD1/PDL1, LAG3, and/or TIM3. In some embodiments,the pulsed nature of neoantigen presentation and priming may allow acheckpoint inhibitor and/or a splicing modulator, ADC, or composition tobe administered less frequently and/or at lower doses. In someembodiments, the pulsed nature of neoantigen presentation may provideone or more treatment benefits for a checkpoint inhibitor (e.g., ananti-CTLA4 antibody such as ipilimumab), as compared to the checkpointinhibitor when administered without concurrent splicing modulator, ADC,or composition treatment, for example, by lowering the potential risk ofadverse reactions often observed with the checkpoint inhibitor'sstandard dosing regimen.

In certain embodiments, the checkpoint inhibitor is an inhibitor of thecytotoxic T-lymphocyte-associated antigen (CTLA4) pathway. CTLA4, alsoknown as CD152, is a protein receptor that downregulates immuneresponses. CTLA4 is constitutively expressed in regulatory T-cells, butonly upregulated in conventional T-cells after activation. As usedherein, the term “CTLA4 inhibitor” is meant to refer to any inhibitor ofCTLA4 and/or the CTLA4 pathway. Exemplary CTLA4 inhibitors include butare not limited to anti-CTLA4 antibodies. CTLA4 blocking antibodies foruse in humans were developed based on the pre-clinical activity seen inmouse models of anti-tumor immunity. Exemplary anti-CTLA4 antibodiesinclude but are not limited to ipilimumab (MDX-010) and tremelimumab(CP-675,206), both of which are fully human. Ipilimumab is an IgG1 witha plasma half-life of approximately 12-14 days; tremelimumab is an IgG2with a plasma half-life of approximately 22 days. See, e.g., Phan et al.(2003) Proc Natl Acad Sci USA. 100:8372-7; Ribas et al. (2005) J ClinOncol. 23:8968-77; Weber et al. (2008) J Clin Oncol. 26:5950-6. In someembodiments, the anti-CTLA4 antibody is ipilimumab.

In certain embodiments, the checkpoint inhibitor is an inhibitor of theprogrammed death-1 (PD1) pathway. The programmed cell death 1 (PD1)pathway represents a major immune control switch which may be engaged bytumor cells to overcome active T-cell immune surveillance. The ligandsfor PD1 (PDL1 and PDL2) are constitutively expressed or can be inducedin various tumors. High expression of PDL1 on tumor cells (and to alesser extent of PDL2) has been found to correlate with poor prognosisand survival in various other solid tumor types. Furthermore, PD1 hasbeen suggested to regulate tumor-specific T-cell expansion in patientswith malignant melanoma. These observations suggest that the PD1/PDL1pathway plays a critical role in the tumor immune evasion and may beconsidered an attractive target for therapeutic intervention. As usedherein, the term “PD1 inhibitor” is meant to refer to any inhibitor ofPD1 and/or the PD1 pathway. Exemplary PD1 inhibitors include but are notlimited to anti-PD1 and anti-PDL1 antibodies. In certain embodiments,the checkpoint inhibitor is an anti-PD1 antibody. Exemplary anti-PD1antibodies include but are not limited to nivolumab and pembrolizumab(MK-3475). Nivolumab, for example, is a fully human immunoglobulin G4(IgG4) PD1 immune checkpoint inhibitor antibody that disrupts theinteraction of the PD1 receptor with its ligands PDL1 and PDL2, therebyinhibiting the cellular immune response (Guo et al. (2017) J Cancer8(3):410-6). In some embodiments, the anti-PD1 antibody is nivolumab.Pembrolizumab, for example, is a potent and highly-selective humanizedmAb of the IgG4/kappa isotype designed to directly block the interactionbetween PD1 and its ligands, PDL1 and PDL2. Pembrolizumab stronglyenhances T lymphocyte immune responses in cultured blood cells fromhealthy human donors, cancer patients, and primates. Pembrolizumab hasalso been reported to modulate the level of interleukin-2 (IL-2), tumornecrosis factor alpha (TNFα), interferon gamma (IFNγ), and othercytokines. Exemplary anti-PDL1 antibodies include but are not limited toatezolizumab, avelumab, and durvalumab. Atezolizumab, for example, is anIgG1 humanized mAb that is reported to block the PD1/PDL1 interaction,by targeting the expressed PDL1 on numerous kinds of malignant cells.This blockage of the PD1/PDL1 pathway may stimulate the immune defensemechanisms against tumors (Abdin et al. (2018) Cancers (Basel)10(2):32). In some embodiments, the anti-PDL1 antibody is atezolizumab.

In certain embodiments, the checkpoint inhibitor is targeted atPD1/PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and/or KIR. In certainembodiments, the checkpoint inhibitor is targeted at CTLA4, OX40, CD40,and/or GITR. In certain embodiments, a checkpoint inhibitor is targetedwith an inhibitory antibody or other similar inhibitory molecule (e.g.,an inhibitory anti-CTLA4 or anti-PD1/PDL1 antibody). In certain otherembodiments, a checkpoint inhibitor is targeted with an agonist for thetarget; examples of this class include the stimulatory targets OX40,CD40, and/or GITR. In some embodiments, the checkpoint inhibitortargeted at OX40, CD40, and/or GITR is an agonist antibody. Agonistantibodies directed against OX40 may have a dual role, inhibitingregulatory T-cell suppression, while enhancing effector T-cellfunctions. Agonist anti-GITR antibodies have also been shown to makeeffector T-cells more resistant to the inhibition induced by regulatoryT-cells (Karaki et al. (2016) Vaccines (Basel) 4(4):37). Likewise,agonist CD40 antibodies demonstrate T-cell-dependent anti-tumoractivity. Activation of CD40 on dendritic cells increasescross-presentation of tumor antigens and consequently the number ofactivated tumor-directed effector T-cells (Ellmark et al. (2015)Oncoimmunol. 4(7):e1011484).

In certain embodiments, the checkpoint inhibitor is targeted at CTLA4(e.g., an anti-CTLA4 antibody). In certain embodiments, targeting CTLA4facilitates priming and activation of naïve T-cells. In certainembodiments, the checkpoint inhibitor is targeted at OX40 (e.g., ananti-OX40 antibody). In certain embodiments, targeting OX40 enhancesexpansion of effector T-cells. In certain embodiments, the checkpointinhibitor is targeted at CD40 (e.g., an anti-CD40 antibody). In certainembodiments, targeting CD40 inhibits “tolerogenic” priming of T-cellsand/or formation of regulatory T-cells. In certain embodiments, thecheckpoint inhibitor is targeted at GITR (e.g., an anti-GITR antibody).In certain embodiments, targeting GITR inhibits activity of regulatoryT-cells. In certain embodiments, the benefit of combination therapy(e.g., the effect on at least one symptom or the risk/rate of diseaseprogression) with a splicing modulator, ADC, or composition and aCTLA4-, OX40-, CD40-, and/or GITR-targeted agent is additive. In someembodiments, the benefit of combination therapy with a splicingmodulator, ADC, or composition and a CTLA4-, OX40-, CD40-, and/orGITR-targeted agent is superadditive (i.e., synergistic).

Checkpoint inhibitor treatment strategies are based on the hypothesisthat treatment facilitates and/or enhances priming of T-cell responsesto weakly or poorly antigenic tumors (e.g., CTLA4) or that treatmentrestores and/or reinvigorates T-cells that respond to tumor antigens,but have become “exhausted” due to the chronic nature of the antigenpresentation (e.g., PD1, PDL1) (Chen and Mellman (2013) Immunity39(1):1-10). Examples of suitable checkpoint inhibition therapies andagents, e.g., anti-PD1, anti-PDL1, or anti-CTLA4 antibodies, are knownin the art. See, e.g., WO 2001/014424 WO 2013/173223, WO 2016/007235.

Combining these primed T-cell responses following checkpoint inhibitortherapy with treatment to induce neoantigens in tumor cells to which theprimed immune system can react may provide beneficial synergy. As thesplicing modulator or ADC-derived neoantigens have not yet beenpresented for T-cell priming, combination with a CTLA4 inhibitor may beparticularly beneficial. In some embodiments, treatment comprisesadministering one or more splicing modulator, ADC, or composition toinduce the production of neoantigens, followed before, concurrently, orthereafter by an initial administration of a CTLA4 inhibitor tostimulate CD8 T-cell priming. In some embodiments, additionaladministrations of an CTLA4 inhibitor are provided to the patient, e.g.,to further stimulate priming and/or activation of neoantigen-reactiveCD8 populations. In some embodiments, additional administrations ofsplicing modulator, ADC, or composition can be given to the patient toincrease neoantigen presentation by the tumor. Repeat administrations ofsplicing modulator, ADC, or composition and checkpoint inhibitor therapycan occur concurrently or in staggered intervals. In some embodiments,treatment further comprises administering a PD1/PDL1 inhibitorco-treatment, e.g., to restore effector function of exhaustedneoantigen-targeted T-cells within the tumor microenvironment.

The terms “combination” or “combination therapy,” as used herein, referto the administration of one or more splicing modulator, ADC, orcomposition together with an additional agent or therapy (e.g., acheckpoint inhibitor, a cytokine or cytokine analog, a neoantigenvaccine, CAR-T), as part of a treatment regimen intended to provide abeneficial (i.e., additive or synergistic) effect from the co-action ofone or more of the administered agents. In some embodiments, thecombination may also include one or more additional agents, includingbut not limited to chemotherapeutic agents, anti-angiogenesis agents,and agents that reduce immune-suppression (e.g., a second checkpointinhibitor). The beneficial effect of the combination includes, but isnot limited to, pharmacokinetic or pharmacodynamic co-action resultingfrom the combination of therapeutic agents. Administration of thesetherapeutic agents in combination typically is carried out over adefined time period (for example, minutes, hours, days, or weeks,depending upon the combination selected).

Administered “in combination” or “co-administration,” as used herein,means that two or more different treatments are delivered to a subjectduring the subject's affliction with a medical condition (e.g., aneoplastic disorder). For example, in some embodiments, the two or moretreatments are delivered after the subject has been diagnosed with adisease or disorder, and before the disease or disorder has been curedor eliminated, or when a subject is identified as being at risk butbefore the subject has developed symptoms of the disease. In someembodiments, the delivery of one treatment is still occurring when thedelivery of the second treatment begins, so that there is overlap. Insome embodiments, the first and second treatment are initiated at thesame time. These types of delivery are sometimes referred to herein as“simultaneous,” “concurrent,” or “concomitant” delivery. In otherembodiments, the delivery of one treatment ends before delivery of thesecond treatment begins. This type of delivery is sometimes referred toherein as “successive” or “sequential” delivery.

In some embodiments, the two treatments (e.g., a splicing modulator,ADC, or composition and a checkpoint inhibitor) are comprised in thesame composition. Such compositions may be administered in anyappropriate form and by any suitable route. In other embodiments, thetwo treatments (e.g., a splicing modulator, ADC, or composition and acheckpoint inhibitor) are administered in separate compositions, in anyappropriate form and by any suitable route. For example, in someembodiments, a composition comprising a splicing modulator or ADC and acomposition comprising a checkpoint inhibitor may be administeredconcurrently or sequentially, in any order at different points in time;in either case, they should be administered sufficiently close in timeso as to provide the desired therapeutic or prophylactic effect.

In embodiments of either simultaneous or sequential delivery, treatmentmay be more effective because of combined administration. In someembodiments, the first treatment is more effective, e.g., an equivalenteffect is seen with less of the first treatment (e.g., with a lowerdose), than would be seen if the first treatment were administered inthe absence of the second treatment. In some embodiments, the firsttreatment is more effective such that the reduction in a symptom, orother parameter associated with the disease or disorder, is greater thanwhat would be observed with the first treatment delivered in the absenceof the second treatment. In other embodiments, an analogous situation isobserved with the second treatment. In some embodiments, the benefit ofcombination therapy (e.g., the effect on at least one symptom or therisk/rate of disease progression) is additive. In some embodiments, thebenefit of combination therapy is superadditive.

In various embodiments, the present disclosure provides a method oftreating a subject having or suspected of having a neoplastic disorderby administering to the subject an effective amount of a splicingmodulator, an ADC, or a composition comprising a splicing modulator orADC; and at least one additional therapy (e.g., a checkpoint inhibitortherapy, a cytokine or cytokine analog, a neoantigen vaccine, CAR-T). Insome embodiments, administration of the splicing modulator, ADC, orcomposition induces at least one neoantigen and/or a T-cell response. Insome embodiments, administration of the splicing modulator, ADC, orcomposition induces a double-stranded RNA immune response. In someembodiments, administration of the splicing modulator, ADC, orcomposition induces immunogenic cell death. In some embodiments, the atleast one additional therapy may comprise at least one, at least two, atleast three, at least four, or at least five additional therapies. Forexample, in some embodiments, a splicing modulator, ADC, or compositionmay be administered in combination with two checkpoint therapies, i.e.,using two different checkpoint inhibitors. In some other embodiments, asplicing modulator, ADC, or composition may be administered incombination with a checkpoint inhibitor therapy and a neoantigenvaccine.

In some embodiments of combination therapy, the administered amount ofthe splicing modulator, antibody-drug conjugate, or composition and/orthe at least one additional therapy is reduced by 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 75%, or 90%, as compared to a standard dosageof the splicing modulator, antibody-drug conjugate, or compositionand/or the at least one additional therapy. In some embodiments, thesplicing modulator, antibody-drug conjugate, or composition and/or theat least one additional therapy is administered at least 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% less frequently, as comparedto a standard dosing regimen of the splicing modulator, antibody-drugconjugate, or composition and/or the at least one additional therapy. Insome embodiments, the administered amount and/or dosage of the splicingmodulator, antibody-drug conjugate, or composition and/or the at leastone additional therapy results in lower systemic toxicity and/orimproved tolerance.

In some embodiments, administration of the splicing modulator,antibody-drug conjugate, or composition is initiated beforeadministration of the at least one additional therapy. In someembodiments, administration of the splicing modulator, antibody-drugconjugate, or composition is initiated after administration of the atleast one additional therapy. In some embodiments, administration of thesplicing modulator, antibody-drug conjugate, or composition is initiatedconcurrently with administration of the at least one additional therapy.

In some embodiments, administration of the splicing modulator,antibody-drug conjugate, or composition is repeated at least once afterinitial administration. In some embodiments, the amount of the splicingmodulator, antibody-drug conjugate, or composition used for repeatedadministration is reduced as compared to the amount used for initialadministration. In some embodiments, the amount of the splicingmodulator, antibody-drug conjugate, or composition used for repeatedadministration is reduced as compared to a standard dosage of thesplicing modulator, antibody-drug conjugate, or composition. In someembodiments, the amount of the splicing modulator, antibody-drugconjugate, or composition used for repeated administration is reduced by10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, as compared toa standard dosage of the splicing modulator, antibody-drug conjugate, orcomposition.

In some embodiments, administration of the at least one additionaltherapy is repeated at least once after initial administration. In someembodiments, the amount of the at least one additional therapy used forrepeated administration is reduced as compared to the amount used forinitial administration. In some embodiments, the amount of the at leastone additional therapy used for repeated administration is reduced ascompared to a standard dosage of the at least one additional therapy. Insome embodiments, the amount of the at least one additional therapy usedfor repeated administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 75%, or 90%, as compared to a standard dosage of the atleast one additional therapy.

In some embodiments, repeated administration of the splicing modulator,antibody-drug conjugate, or composition is concurrent with repeatedadministration of the at least one additional therapy. In someembodiments, repeated administration of the splicing modulator,antibody-drug conjugate, or composition is sequential or staggered withrepeated administration of the at least one additional therapy.

In various embodiments, the present disclosure provides a method oftreating a subject having or suspected of having a neoplastic disorderby administering to the subject an effective amount of a splicingmodulator, an ADC, or a composition comprising a splicing modulator orADC; and a checkpoint inhibitor therapy. In some embodiments, thecheckpoint inhibitor therapy comprises administering at least onecheckpoint inhibitor. In some embodiments, the subject is intolerant,non-responsive, or poorly responsive to the at least one checkpointinhibitor when administered alone. In some embodiments, a subject may beconsidered non-responsive or poorly responsive to the at least onecheckpoint inhibitor as determined using, e.g., the immune-relatedResponse Criteria (irRC) and/or the immune-related Response EvaluationCriteria in Solid Tumors (irRECIST). See, e.g., Wolchok et al. (2009)Clin Cancer Res. 15(23):7412-20; Bohnsack et al. “Adaptation of theImmune-Related Response Criteria:irRECIST” (Abstract 4958) ESMO 2014.Exemplary criteria may include those used in the art to define whentumors in cancer patients improve (“respond”), remain the same(“stabilize”), or worsen (“progress”) during treatment, when thetreatment being evaluated is an immune-oncology drug (e.g., a checkpointinhibitor). In some embodiments, a subject may be considered intolerantto the at least one checkpoint inhibitor if the subject presents withone or more than one adverse (grade 2+) event identified for therespective checkpoint inhibitor (e.g., ipilimumab). In some embodiments,for example, a subject may be considered intolerant to ipilimumabtreatment if the subject presents with one or more adverse eventsselected from enterocolitis, hepatitis, dermatitis (including toxicepidermal necrolysis), neuropathy, and endocrinopathy (Yervoy®(ipilimumab) FDA Label Supplement, 2018).

In some embodiments, the checkpoint inhibitor is targeted at PD1/PDL1,CTLA4, OX40, CD40, LAG3, TIM3, GITR, and/or KIR. In some embodiments,the checkpoint inhibitor is targeted at CTLA4, OX40, CD40, and/or GITR.In some embodiments, the checkpoint inhibitor is targeted with aninhibitory antibody or other similar inhibitory molecule. In some otherembodiments, the checkpoint inhibitor is targeted with an agonistantibody or other similar agonist molecule. In some embodiments, thecheckpoint inhibitor comprises a cytotoxic T-lymphocyte-associatedantigen 4 pathway (CTLA4) inhibitor. In some embodiments, the CTLA4inhibitor is an anti-CTLA4 antibody. In some embodiments, the anti-CTLA4antibody is ipilimumab. In some embodiments, the checkpoint inhibitorcomprises a programmed death-1 pathway (PD1) inhibitor. In someembodiments, the PD1 inhibitor is an anti-PD1 antibody. In someembodiments, the anti-PD1 antibody is nivolumab. In some embodiments,the PD1 inhibitor is an anti-PDL1 antibody. In some embodiments, theanti-PDL1 antibody is atezolizumab. In some embodiments, the checkpointinhibitor comprises a CTLA4 inhibitor and a PD1 inhibitor. In someembodiments, the checkpoint inhibitor is targeted at OX40. In someembodiments, the checkpoint inhibitor is targeted at CD40. In someembodiments, the checkpoint inhibitor is targeted at GITR. In someembodiments, the benefit of combination therapy (e.g., the effect on atleast one symptom or the risk/rate of disease progression) with asplicing modulator, ADC, or composition and a checkpoint inhibitor(e.g., a CTLA4-, PD1/PDL1-, OX40-, CD40-, and/or GITR-targeted antibodyor molecule) is additive. In some embodiments, the benefit ofcombination therapy with a splicing modulator, ADC, or composition and acheckpoint inhibitor (e.g., a CTLA4-, PD1/PDL1, OX40-, CD40-, and/orGITR-targeted antibody or molecule) is superadditive (i.e.,synergistic).

In various embodiments, the present disclosure provides a method oftreating a subject having or suspected of having a neoplastic disorderby administering to the subject an effective amount of a splicingmodulator, an ADC, or a composition comprising a splicing modulator orADC; and a cytokine or cytokine analog therapy. In some embodiments, thecytokine or cytokine analog therapy comprises administering at least onecytokine or cytokine analog. In some embodiments, the subject isintolerant, non-responsive, or poorly responsive to the at least onecytokine or cytokine analog when administered alone.

In some embodiments, the cytokine or cytokine analog comprises a T-cellenhancer. In some embodiments, the cytokine or cytokine analog comprisesIL-2, IL-10, IL-12, IL-15, IFNγ, and/or TNFα. In some embodiments, thecytokine or cytokine analog comprises IL-2, IL-10, IL-12, and/or IL-15.In some embodiments, administering the cytokine or cytokine analogenhances T-cell priming following administration of a splicingmodulator, antibody-drug conjugate, or composition due to induction andpresentation of neoantigens.

In some embodiments, the cytokine or cytokine analog comprises IL-2. Insome embodiments, IL-2 boosts signals to effector cells promoting theirexpansion (Rosenberg (2014) J Immunol. 192(12):5451-8). In someembodiments, the cytokine or cytokine analog comprises IL-10. In someembodiments, IL-10 boosts CD8+ T-cell priming and activation (Mumm etal. (2011) Cancer Cell 20(6):781-96). In some embodiments, the cytokineor cytokine analog comprises IL-12. In some embodiments, IL-12 links theinnate and adaptive immune responses to boost antigen-specific primingand targeting (Tugues et al. (2015) Cell Death Differ. 22(2):237-46). Insome embodiments, the cytokine or cytokine analog comprises IL-15. Insome embodiments, IL-15 boosts T-effector (CD8) cell priming and/oractivation. In some embodiments, the cytokine or cytokine analogcomprises IFNγ. In some embodiments, IFNγ supplements T-effector cellsecretion of IFNγ. In some embodiments, the cytokine or cytokine analogcomprises TNFα. In some embodiments, TNFα supplements T-effector cellsecretion of TNFα.

In some embodiments, an initial dose of a splicing modulator, ADC, orcomposition is administered to a subject to trigger aberrant splicingand production of neoantigen peptides. After a period to allow forprotein production and antigen presentation, in some embodiments, thesubject is then administered an initial dose of a cytokine or cytokineanalog to boost and/or enhance effector T-cell priming and expansion. Insome embodiments, the wait period between doses of splicing modulator,ADC, or composition and cytokine or cytokine analog is about 2, about 3,about 4, about 5, about 6, or about 7 days. In some embodiments, thewait period is between about 3 days and about 5 days. In someembodiments, the cytokine or cytokine analog is IL-2, IL-10, IL-12,IL-15, IFNγ, and/or TNFα. In some embodiments, the combinationtherapeutic benefit of a splicing modulator, ADC, or composition and acytokine or cytokine analog may be additive or superadditive.

In some other embodiments, an initial dose of a cytokine or cytokineanalog is administered to a subject to boost and/or enhance effectorT-cell priming and expansion. After a wait period, in some embodiments,the subject is then administered an initial dose of a splicingmodulator, ADC, or composition to trigger aberrant splicing andproduction of neoantigen peptides. In some embodiments, the wait periodbetween doses of cytokine or cytokine analog and splicing modulator,ADC, or composition is about 2, about 3, about 4, about 5, about 6, orabout 7 days. In some embodiments, the wait period is between about 3days and about 5 days. In some embodiments, the cytokine or cytokineanalog is IL-2, IL-10, IL-12, IL-15, IFNγ, and/or TNFα. In someembodiments, the combination therapeutic benefit of a cytokine orcytokine analog and a splicing modulator, ADC, or composition may beadditive or superadditive.

In some embodiments, after a period to allow for T-cell priming andexpansion, the subject is then administered a second or subsequent doseof the splicing modulator, ADC, or composition to triggerre-presentation of neoantigen peptides. In some embodiments, the waitperiod between an initial dose of a cytokine or cytokine analog and asecond or subsequent dose of a splicing modulator, ADC, or compositionis about 2, about 3, about 4, or about 5 weeks. In some embodiments, thewait period is about 3 weeks. In some embodiments, subsequent doses ofthe cytokine or cytokine analog may be administered, e.g., interspersedbetween subsequent doses of the splicing modulator, ADC, or composition.Following a second or subsequent dose of the splicing modulator, ADC, orcomposition, in some embodiments, the immune system may engage with theneoantigen-presenting tumor cells and/or elicit tumor cell killing. Insome embodiments, dosing of the splicing modulator, ADC, or compositionfollowing this exemplary initial treatment regimen can be pulsatile,i.e., the splicing modulator, ADC, or composition may be dosed atprolonged intervals (e.g., about every 4 weeks, about every 5 weeks,about every 6 weeks) to allow for antigen presentation, T-cellengagement and/or tumor cell killing, and/or recovery of the memoryT-cell population.

In some embodiments, the subject has a non-synonymous mutational burdenof about 150 mutations or less. In some embodiments, the subject has anon-synonymous mutational burden of about 100 mutations or less. In someembodiments, the subject has a non-synonymous mutational burden of about50 mutations or less. In some embodiments, the subject has or issuspected of having a neoplastic disorder, e.g., a hematologicalmalignancy or a solid tumor. In some embodiments, the hematologicalmalignancy is selected from a B-cell malignancy, a leukemia, a lymphoma,and a myeloma. In some embodiments, the hematological malignancy isselected from acute myeloid leukemia and multiple myeloma. In someembodiments, the solid tumor is selected from breast cancer, gastriccancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer,salivary duct carcinoma, melanoma, colon cancer, cervical cancer,pancreatic cancer, kidney cancer, colorectal cancer, and esophagealcancer. In some embodiments, the solid tumor is selected fromHER2-positive breast cancer, gastric adenocarcinoma, prostate cancer,and osteosarcoma.

Combination of Splicing Modulator/ADC and Neoantigen Vaccine:

In various embodiments, a patient having a cancer as described hereincan be treated with a combination of a splicing modulator, ADC, orcomposition and a neoantigen vaccine. Without being bound by theory,vaccines, used alone or in combination with immune checkpoint inhibitor(ICI) molecules, have shown promise in early trials (Ott et al. (2017)Nature 547(7662):217-21; Sahin et al. (2017) Nature 547(7662):222-6),but generally require sequencing of patient tumor mutations (Ott et al.(2017) Nature 547(7662):217-21; Aldous and Dong (2018) Bioorg. Med.Chem. 26(10):2842-9). As such, vaccines are often dependent onsufficient numbers of non-synonymous mutations that are antigenic. Ingeneral, tumors with very low mutation burden provide few candidateantigens, and those with rapid growth provide limited time to identifyand produce patient-specific vaccines.

To date, attempts to develop vaccines that would be broadly immunogenicacross a large percentage of patients have focused on proteins that areeither frequently mutated, ectopically overexpressed, or amplified,and/or that exist as “self” proteins within the organism. In addition,these proteins are often expressed in immunologically restricted tissues(e.g., neuronal markers expressed in neuroendocrine tumor types), whileothers may be normally expressed during embryogenesis (e.g., oncofetalantigens). Thus, utility of vaccines using such proteins as antigens isoften limited to specific tumor lineages or subsets where one or more ofthe antigens are presented. Vaccine utility would also need to beconfirmed by sequencing of patient tumor samples, which can betime-consuming.

Moreover, if these antigens exist as “self” proteins, the immune systemwould likely be primed to recognize these as “self” and thus, notrespond. Or, alternatively, if the immune system is able to mount aneffector response to these antigens, it may lead to on-target sideeffects in tissues where the antigen may be expressed. In both of thesecases, one of the key challenges is that most antigenic peptides arederived from “passenger” genes (i.e., genes that are mutated oramplified in the course of tumorigenesis, but that do not play acritical role in the continued survival or proliferation of the tumoritself). As such, these genes may be silenced without significantconsequence to the tumor progression, and thus would allow a tumor to“escape” an immune response against these antigens. Without wishing tobe bound by theory, this mechanism may play a role in tumor evolution,where random mutations that are strongly antigenic are often “selectedagainst” by the tumor during the early stages of tumorigenesis (Dunn etal. (2004) Annu. Rev. Immunol. 22:329-60).

In addition, certain evidence also indicates that chronic antigenpresentation and immune stimulation may lead to immune cell anergy andexhaustion (Pardoll (2012) Nat. Rev. Cancer 12(4):252-64). Thesephenotypes underlie the therapeutic rationale behind current ICItreatments, as ICI has been shown to either repress the exhausted immunecell phenotype (α-PD1/PD-L1) or to facilitate additional immune cellresponses (α-CTLA4). Notably, with α-CTLA4 therapy, a certain subset ofpatients have been reported to exhibit severe immune-related adverseevents that may be ascribed to the promotion of T-cell activation and abreak of the immune tolerance mechanisms that restrain self-reactiveimmune responses.

Both of these approaches (i.e., triggering or enhancing de novo immuneresponses to neoantigens or derepressing the anergy or exhaustion ofexisting immune responses) are linked to a chronic immune activation. Assuch, these approaches are sensitive to anergy, editing, and othertumor-mediated mechanisms designed to suppress immune engagement.

In contrast, treatment with a splicing modulator, ADC, or compositiondisclosed herein may induce an immune response to novel sequencesrepresenting neoantigens. In some embodiments, presentation ofneoantigens provides the adaptive immune system with more divergenttargets with which to engage and activate. In some embodiments, theability of a splicing modulator, ADC, or composition to acutely inducealternative splicing and the resulting neoantigens may reduce the riskof immune system fatigue due to chronic exposure to mutation-drivenneoantigens and/or limit the ability of tumor cells to adapt to evadetherapy. In some embodiments, administering a splicing modulator, ADC,or composition in combination with a neoantigen vaccine enhances theimmune response to the neoantigens produced by the splicing modulator,ADC, or composition. In some embodiments, the splicing modulator, ADC,or composition is administered before, during, or after vaccination. Insome embodiments, the splicing modulator, ADC, or composition and/orvaccine may be administered once or more than once during the course oftreatment. In some embodiments, the vaccine is administered once and thesplicing modulator, ADC, or composition is administered more than onceduring the course of treatment. In some embodiments, the vaccine isadministered once and then one or more boosters are administered duringthe course of treatment.

As used herein, the term “neoantigen vaccine” refers to a pooled sampleof one or more immunogenic neoantigen peptides or mRNAs, for example atleast two, at least three, at least four, at least five, or moreneoantigen peptides. The term “vaccine” refers to a composition forgenerating immunity for the prophylaxis and/or treatment of a disease(e.g., a neoplastic disorder, e.g., a hematological malignancy or solidtumor). Accordingly, vaccines are medicaments which comprise immunogenicagents and are intended to be used in humans or animals for generatingspecific immune defenses and protective substances after vaccination. Aneoantigen vaccine can additionally include a pharmaceuticallyacceptable carrier, diluent, excipient, and/or adjuvant.

As used herein, the term “immunogenic” refers to any agent orcomposition that can elicit an immune response, e.g., a T-cell response.The immune response can be antibody- or cell-mediated, or both.

In some embodiments, a patient is given a splicing modulator, ADC, orcomposition and then given a peptide or mRNA vaccine of known neoantigento enhance immune response to the neoantigens produced by the splicingmodulator, ADC, or composition. In some other embodiments, a patient isgiven a splicing modulator, ADC, or composition and screened forneoantigens produced by the treatment. Subsequently, one or more ofthose neoantigens are used to create a personalized vaccine that isgiven to the patient. In either of these embodiments, the splicingmodulator, ADC, or composition and/or peptide or mRNA vaccine may beadministered to the patient once or repeatedly.

In various embodiments, a suitable neoantigen for a vaccine can beidentified by screening a panel of transcripts with altered splicing androbust expression from one or more tissue samples in a patient (e.g.,from a tumor biopsy). In some embodiments, variant protein sequences areidentified in the screened sample based on translation across theaberrantly spliced mRNA junction while retaining portions of the proteinsequence (up to 12 amino acids) flanking the junction-spanning aminoacid changes. In some embodiments, these junction-spanning peptidefragments are scanned for high affinity binding to MHC1 alleles, e.g.,using a tool such as NetMHC1 (Nielsen et al. (2003) Protein Sci12(5):1007-17; Andreatta and Neilsen (2016) Bioinformatics 32(4):511-7).These results allow for filtering of the neopeptides to those that arepredicted high affinity binders for a unique patient HLA allele makeupas well as assembly of pools of neopeptides predicted to be broadlybinding to HLA alleles that are present with high frequencies indifferent populations (Maiers et al. (2007) Hum Immunol 68(9):779-88).In various embodiments, the identified neopeptides are then formulatedas a vaccine, e.g., by conjugation to a suitable carrier or adjuvant(Ott et al. (2017) Nature 547(7662):217-21), or for delivery as an mRNA(Sahin et al. (2017) Nature 547(7662):222-6).

In some embodiments, the selected neoantigen is based on a screen of anindividual patent's tumor response to the splicing modulator, ADC, orcomposition to identify one or more neoantigens resulting from treatmentto use in subsequent vaccination. In other embodiments, a neoantigen ischosen, e.g., based on screening a panel of samples from differentpatients to identify common neoantigens produced by the splicingmodulator, ADC, or composition and then used as a universal vaccine forfuture patients.

Without being bound by theory, in some embodiments, use of a universalneoantigen vaccine would avoid the need to sequence and analyze theunique mutation status of each patient's tumor because the chosenneoantigens are not dependent on tumor mutation but rather mimic aneoantigen produced by a splicing modulator, ADC, or composition andgenerally recognized by the body as foreign. In addition, in someembodiments, use of a neoantigen vaccine may be particularly effectivesince a patient's tumor cells may be more likely to mutate away fromproducing one or more neoantigens that are dependent on tumor mutation,as compared to those that mimic a neoantigen produced by a splicingmodulator, ADC, or composition. This may allow for the formulation of abulk vaccine that would be broadly immunogenic across a large percentageof patients, expediting the initiation of a treatment regime. Patientsmay be vaccinated according to the schedules outlined herein and, priorto following completion of the vaccination, could be further treatedwith a splicing modulator, ADC, or composition, e.g., to induceexpression of the neoantigen peptides. In some embodiments, patients maybe administered a splicing modulator, ADC, or composition before, at thesame time as, or after vaccination. In some embodiments, patients areadministered a splicing modulator, ADC, or composition, screened for oneor more neoantigens found in a panel of universal neoantigens, andvaccinated with a universal neoantigen vaccine comprising at least oneuniversal neoantigen identified in the subject. In some embodiments,patients may be administered a splicing modulator, ADC, or compositiononce or more than once after vaccination. Splicing modulator or ADC orcomposition and/or vaccine may be administered once or more than onceduring the course of treatment.

In various embodiments, a vaccine may comprise one or more than oneneoantigen peptide or mRNA. In various embodiments, a vaccine maycomprise one or more than one long neoantigen peptide. Such “long”neoantigen peptides, in various embodiments, undergo efficientinternalization, processing, and cross-presentation in professionalantigen-presenting cells such as dendritic cells. Similarly, longvaccine peptides have been shown, in other contexts, to induce cytotoxicT-cells in humans (Melief and van der Burg (2008) Nat Rev Cancer8(5):351-60). In various embodiments, a neoantigen peptide is extendedto comprise the neoantigen peptide sequence itself in addition toflanking amino acid sequences. In various embodiments, the extendedpeptide sequence facilitates the uptake of protein by antigen-presentingcells, e.g., dendritic cells. In various embodiments, the extendedpeptide sequence enables efficient antigen presentation and T-cellpriming in models with different HLA isotypes. In various embodiments, alonger neoantigen peptide and/or extended peptide sequence exhibitsincreased uptake by antigen-presenting cells (e.g., dendritic cells),increased antigen presentation, and/or increased T-cell priming, ascompared to a shorter neoantigen peptide and/or shorter peptide sequence(e.g., a peptide sequence less than about 10 or less than about 5 aminoacids in length). In some embodiments, a long neoantigen peptide rangesfrom about 5 to about 50 amino acids in length. In some embodiments, along neoantigen peptide ranges from about 10 to about 50 amino acids inlength. In some embodiments, a long neoantigen peptide ranges from about10 to about 35 amino acids in length. In some embodiments, a longneoantigen peptide ranges from about 15 to about 25 amino acids inlength. Amino acid sequences of exemplary long neoantigen peptides areset forth in Table 21.

As used herein, a neoantigen peptide or mRNA vaccine encompasses using afragment of a neoantigen peptide or its encoding mRNA, so long as thatfragment retains immunogenic potential.

In some embodiments, a neoantigen vaccine comprises at least oneneoantigen peptide. In some embodiments, a neoantigen vaccine comprisesat least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 12, at least 15, or atleast 20 neoantigen peptides. In some embodiments, the neoantigenpeptide(s) range from about 5 to about 50 amino acids in length. In someembodiments, the neoantigen peptide(s) range from about 10 to about 50amino acids in length. In some embodiments, the neoantigen peptide(s)range from about 10 to about 35 amino acids in length. In someembodiments, the neoantigen peptide(s) range from about 15 to about 25amino acids in length.

In various embodiments, the present disclosure provides a method oftreating a subject having or suspected of having a neoplastic disorderby administering to the subject an effective amount of a splicingmodulator, an ADC, or a composition comprising a splicing modulator orADC; and a neoantigen vaccine. A neoantigen vaccine may be, e.g., apeptide or mRNA neoantigen vaccine. In some embodiments, the splicingmodulator, ADC, or composition is administered before administration ofthe neoantigen vaccine. In some embodiments, the splicing modulator,ADC, or composition is administered after administration of theneoantigen vaccine. In some embodiments, the splicing modulator, ADC, orcomposition is administered concurrently with administration of theneoantigen vaccine. In some embodiments, administration of the splicingmodulator, ADC, or composition is repeated at least once after initialadministration. In some embodiments, the amount of the splicingmodulator, ADC, or composition used for repeated administration isreduced as compared to the amount used for initial administration.

In various embodiments, the present disclosure further provides acombination comprising a splicing modulator, an ADC, or a compositioncomprising a splicing modulator or ADC; and a neoantigen vaccine (e.g.,a universal neoantigen vaccine) for use in treating a subject having orsuspected of having a neoplastic disorder. In some embodiments, theneoantigen vaccine is a peptide or mRNA neoantigen vaccine. In someembodiments, the combination further comprises at least one additionaltherapy. In some embodiments, the at least one additional therapycomprises at least one, at least two, at least three, at least four, orat least five additional therapies.

In various embodiments, the present disclosure further provides a methodof treating a subject having or suspected of having a neoplasticdisorder by (a) administering to the subject an effective amount of asplicing modulator, an ADC, or a composition comprising a splicingmodulator or ADC; (b) detecting one or more neoantigens in the subjectafter administration of the splicing modulator, ADC, or composition; (c)comparing the one or more neoantigens to a panel of universalneoantigens; and (d) administering to the subject a universal neoantigenvaccine comprising at least one universal neoantigen present in thesubject. In some embodiments, the universal neoantigen vaccine isadministered alone or in combination with at least one additionaltherapy. In some embodiments, the at least one additional therapycomprises at least one, at least two, at least three, at least four, orat least five additional therapies.

In some embodiments, the at least one additional therapy comprisesrepeated administration of the splicing modulator, ADC, or composition.In some embodiments, repeated administration of the splicing modulator,ADC, or composition is initiated before administration of the universalneoantigen vaccine. In some embodiments, repeated of the splicingmodulator, ADC, or composition is initiated after administration of theuniversal neoantigen vaccine. In some embodiments, repeatedadministration of the splicing modulator, ADC, or composition isinitiated concurrently with administration of the universal neoantigenvaccine. In some embodiments, the amount of the splicing modulator, ADC,or composition used for repeated administration is reduced as comparedto the amount used for initial administration. In some embodiments, theamount of the splicing modulator, ADC, or composition used for theinitial and/or repeated administration is reduced as compared to astandard dosage of the splicing modulator, ADC, or composition when usedwithout a vaccine treatment. In some embodiments, the amount of thesplicing modulator, ADC, or composition used for initial and/or repeatedadministration is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 75%, or 90%, as compared to a standard dosage of the splicingmodulator, ADC, or composition.

In some embodiments, the at least one additional therapy comprisesadministering a checkpoint inhibitor (e.g., any of the exemplarycheckpoint inhibitors described herein). In some embodiments,administration of the checkpoint inhibitor is initiated beforeadministration of the universal neoantigen vaccine and/or repeatedadministration of the splicing modulator, ADC, or composition. In someembodiments, administration of the checkpoint inhibitor is initiatedafter administration of the universal neoantigen vaccine and/or repeatedof the splicing modulator, ADC, or composition. In some embodiments,administration of the checkpoint inhibitor is initiated concurrentlywith administration of the universal neoantigen vaccine and/or repeatedadministration of the splicing modulator, ADC, or composition. In someembodiments, administration of the checkpoint inhibitor is repeated atleast once after initial administration. In some embodiments, the amountof the checkpoint inhibitor used for repeated administration is reducedas compared to the amount used for initial administration. In someembodiments, the amount of the checkpoint inhibitor used for repeatedadministration is reduced as compared to a standard dosage of thecheckpoint inhibitor. In some embodiments, the amount of the checkpointinhibitor used for repeated administration is reduced by 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, as compared to a standarddosage of the checkpoint inhibitor. In some embodiments, the subject isintolerant, non-responsive, or poorly responsive to the checkpointinhibitor when administered alone.

Also provided herein, in various embodiments, are neoantigen vaccinescomprising at least one neoantigen peptide or at least one neoantigenmRNA. In some embodiments, a neoantigen vaccine comprises at least oneneoantigen peptide. In some other embodiments, a neoantigen vaccinecomprises at least one neoantigen mRNA.

Also provided herein, in various embodiments, are kits comprising asplicing modulator, an ADC, or a composition comprising a splicingmodulator or ADC; and a neoantigen vaccine (e.g., a universal neoantigenvaccine). In some embodiments, the neoantigen vaccine is a peptide ormRNA neoantigen vaccine. In some embodiments, the kit further comprisesone or more additional components, including but not limited to:instructions for use; other agents, e.g., one or more additionaltherapeutic agents; devices, containers, or other materials forpreparing the splicing modulator, ADC, composition, and/or neoantigenvaccine for therapeutic administration; pharmaceutically acceptablecarriers; and devices, containers, or other materials for administeringthe splicing modulator, ADC, composition, and/or neoantigen vaccine to apatient. Instructions for use can include guidance for therapeuticapplications including suggested dosages and/or modes of administration,e.g., in a patient having or suspected of having a neoplastic disorder.In various embodiments, the kit further contains instructions fortherapeutic use, e.g., use of the splicing modulator, ADC, orcomposition, and the neoantigen vaccine to treat or prevent a neoplasticdisorder in a patient. In various embodiments, the kit further containsat least one additional therapeutic agent (e.g., for administeringtogether with the splicing modulator, ADC, or composition, and theneoantigen vaccine, e.g., a checkpoint inhibitor). In variousembodiments, the splicing modulator, ADC, composition, and/or neoantigenvaccine is formulated as a pharmaceutical composition.

In some embodiments of the methods and compositions disclosed herein,the neoantigen vaccine comprises at least one neoantigen peptide. Insome embodiments, the at least one neoantigen peptide ranges from about10 to about 50 amino acids in length. In some embodiments, the at leastone neoantigen peptide ranges from about 10 to about 35 amino acids inlength. In some embodiments, the at least one neoantigen peptide rangesfrom about 15 to about 25 amino acids in length.

In some embodiments, the at least one neoantigen peptide comprises oneor more than one neoantigen sequence. In some embodiments, theneoantigen sequence comprises an amino acid sequence of any one of SEQID NOs: 37-65. In some embodiments, the neoantigen sequence comprises anamino acid sequence of SEQ ID NO: 37. In some embodiments, theneoantigen sequence comprises an amino acid sequence of SEQ ID NO: 39.In some embodiments, the neoantigen sequence comprises an amino acidsequence of any one of SEQ ID NOs: 46-49.

In some other embodiments, the neoantigen sequence comprises an aminoacid sequence of any one of SEQ ID NOs: 66-93, or an antigenic portionof any one of SEQ ID NOs: 66-93. In some embodiments, the neoantigensequence comprises an amino acid sequence of SEQ ID NO: 66, or anantigenic portion of SEQ ID NO: 66. In some embodiments, the neoantigensequence comprises an amino acid sequence of any one of SEQ ID NOs:74-77, or an antigenic portion of any one of SEQ ID NOs: 74-77. In someembodiments, the neoantigen sequence and/or antigenic portion rangesfrom about 10 to about 50 amino acids in length. In some embodiments,the neoantigen sequence and/or antigenic portion ranges from about 10 toabout 35 amino acids in length. In some embodiments, the neoantigensequence and/or antigenic portion ranges from about 15 to about 25 aminoacids in length. In some embodiments, the neoantigen sequence and/orantigenic portion ranges from about 10 to about 20 amino acids inlength. In some embodiments, the neoantigen sequence and/or antigenicportion does not exclusively overlap or consist of the canonical peptidesequence (e.g., any of the exemplary canonical peptide sequencesunderlined in Table 21).

In some embodiments, the neoantigen sequence is a neoantigen sequencespecific to the subject. In some embodiments, the neoantigen sequence isa personalized neoantigen vaccine for the subject. In some embodiments,the neoantigen sequence is capable of binding to at least one HLA alleleexpressed in the subject.

In some other embodiments, the neoantigen sequence is a universalneoantigen sequence. In some embodiments, the neoantigen sequence is auniversal neoantigen vaccine. In some embodiments, the neoantigensequence is capable of binding to at least one HLA allele expressed inat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% of subjects in a population ofsubjects suffering from the neoplastic disorder. In some embodiments,the neoantigen sequence is capable of eliciting a T-cell responseagainst a tumor present in at least 1%, at least 5%, or at least 10% ofa population of subjects suffering from the neoplastic disorder.

In some embodiments, the neoantigen sequence has been identified bysequencing at least one neoantigen peptide induced in the subject byadministering an effective amount of the splicing modulator,antibody-drug conjugate, or composition. In some embodiments, the atleast one neoantigen peptide comprises a neoantigen sequence induced bycontacting a neoplastic cell with an effective amount of the splicingmodulator, antibody-drug conjugate, or composition. In some embodiments,the neoplastic cell is present in an in vitro cell culture. In someembodiments, the neoplastic cell is obtained from the subject. In someembodiments, the neoplastic cell is present in the subject.

In some embodiments, the neoantigen vaccine comprises at least oneneoantigen peptide or mRNA and a pharmaceutically acceptable carrier. Invarious embodiments, a neoantigen peptide or mRNA can be linked to asuitable carrier to help elicit an immune response. Exemplary carriersfor linking to immunogenic agents (e.g., a neoantigen peptide or mRNA)include serum albumins, keyhole limpet hemocyanin, immunoglobulinmolecules, thyroglobulin, ovalbumin, tetanus toxoid, or a toxoid fromother pathogenic bacteria, such as diphtheria, E. coli, cholera, or H.pylori, or an attenuated toxin derivative. Other carriers forstimulating or enhancing an immune response include cytokines such asIL-1, IL-1α and β peptides, IL-2, γINF, IL-10, GM-CSF, and chemokines,such as M1P1α and β and RANTES. Immunogenic agents can also be linked topeptides that enhance transport across tissues, as described, e.g., inWO 97/17613 and WO 97/17614. In some embodiments, the pharmaceuticallyacceptable carrier is selected from a peptide, a serum albumin, akeyhole limpet hemocyanin, an immunoglobulin, a thyroglobulin, anovalbumin, a toxoid or an attenuated toxoid derivative, a cytokine, anda chemokine.

In some embodiments, the neoantigen peptide or mRNA may be linked to thepharmaceutically acceptable carrier. Immunogenic agents can be linked tocarriers by chemical crosslinking. Techniques for linking an immunogenicpeptide to a carrier include the formation of disulfide linkages usingN-succinimidyl-3-(2-pyridyl-thio) propionate (SPDP) and succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptidelacks a sulfhydryl group, this can be provided by addition of a cysteineresidue). These reagents create a disulfide linkage between themselvesand peptide cysteine resides on one protein and an amide linkage throughthe epsilon-amino on a lysine, or other free amino group in other aminoacids. A variety of such disulfide/amide-forming agents are described inJansen et al. ((1982) Immun Rev. 62:185). Other bifunctional couplingagents form a thioether rather than a disulfide linkage. Many of thesethioether-forming agents are commercially available and include reactiveesters of 6-maleimidocaproic acid, 2-bromoacetic acid, and 2-iodoaceticacid, 4-(N-maleimido-methyl)cyclohexane-1-carboxylic acid. The carboxylgroups can be activated by combining them with succinimide or1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. In some embodiments,the neoantigen peptide and the pharmaceutically acceptable carrier arecovalently attached via a linker.

Neoantigen and other such immunogenic peptides can also be expressed asfusion proteins with carriers. The immunogenic peptide can be linked atthe amino terminus, the carboxyl terminus, or at a site anywhere withinthe peptide (internally) to the carrier. In some embodiments, multiplerepeats of the immunogenic peptide can be present in the fusion protein.In some embodiments, the neoantigen peptide and the pharmaceuticallyacceptable carrier are expressed as a fusion protein.

In some embodiments, the neoantigen vaccine comprises at least oneneoantigen peptide or its encoding mRNA and a pharmaceuticallyacceptable diluent. In some embodiments, the neoantigen vaccinecomprises at least one neoantigen peptide or its encoding mRNA and apharmaceutically acceptable adjuvant (e.g., an adjuvant as describedherein).

In some embodiments of the methods and compositions disclosed herein,the neoantigen vaccine comprises at least one neoantigen mRNA. In someembodiments, the at least one neoantigen mRNA encodes one or more thanone neoantigen sequence. In some embodiments, the neoantigen sequencecomprises an amino acid sequence of any one of SEQ ID NOs: 37-65. Insome embodiments, the neoantigen sequence comprises an amino acidsequence of SEQ ID NO: 37. In some embodiments, the neoantigen sequencecomprises an amino acid sequence of SEQ ID NO: 39. In some embodiments,the neoantigen sequence comprises an amino acid sequence of any one ofSEQ ID NOs: 46-49.

In some other embodiments, the neoantigen sequence comprises an aminoacid sequence of any one of SEQ ID NOs: 66-93, or an antigenic portionof any one of SEQ ID NOs: 66-93. In some embodiments, the neoantigensequence comprises an amino acid sequence of SEQ ID NO: 66, or anantigenic portion of SEQ ID NO: 66. In some embodiments, the neoantigensequence comprises an amino acid sequence of any one of SEQ ID NOs:74-77, or an antigenic portion of any one of SEQ ID NOs: 74-77. In someembodiments, the neoantigen sequence and/or antigenic portion rangesfrom about 10 to about 50 amino acids in length. In some embodiments,the neoantigen sequence and/or antigenic portion ranges from about 10 toabout 35 amino acids in length. In some embodiments, the neoantigensequence and/or antigenic portion ranges from about 15 to about 25 aminoacids in length. In some embodiments, the neoantigen sequence and/orantigenic portion ranges from about 10 to about 20 amino acids inlength. In some embodiments, the neoantigen sequence and/or antigenicportion does not exclusively overlap or consist of the canonical peptidesequence (e.g., any of the exemplary canonical peptide sequencesunderlined in Table 21).

In some embodiments, the neoantigen sequence is a neoantigen sequencespecific to the subject. In some embodiments, the neoantigen sequence isa personalized neoantigen vaccine for the subject. In some embodiments,the neoantigen sequence is capable of binding to at least one HLA alleleexpressed in the subject.

In some other embodiments, the neoantigen sequence is a universalneoantigen sequence. In some embodiments, the neoantigen sequence is auniversal neoantigen vaccine. In some embodiments, the neoantigensequence is capable of binding to at least one HLA allele expressed inat least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, or at least 45% of subjects in a population ofsubjects suffering from the neoplastic disorder. In some embodiments,the neoantigen sequence is capable of eliciting a T-cell responseagainst a tumor present in at least 1%, at least 5%, or at least 10% ofa population of subjects suffering from the neoplastic disorder.

In some embodiments, the neoantigen sequence has been identified bysequencing at least one neoantigen mRNA induced in the subject byadministering an effective amount of the splicing modulator,antibody-drug conjugate, or composition. In some embodiments, the atleast one neoantigen mRNA encodes a neoantigen sequence induced bycontacting a neoplastic cell with an effective amount of the splicingmodulator, antibody-drug conjugate, or composition. In some embodiments,the neoplastic cell is present in an in vitro cell culture. In someembodiments, the neoplastic cell is obtained from the subject. In someembodiments, the neoplastic cell is present in the subject.

In some embodiments, the neoantigen vaccine comprises at least oneneoantigen mRNA and a pharmaceutically acceptable carrier. In someembodiments, the at least one neoantigen mRNA is linked to thepharmaceutically acceptable carrier. In some embodiments, thepharmaceutically acceptable carrier is selected from a peptide, a serumalbumin, a keyhole limpet hemocyanin, an immunoglobulin, athyroglobulin, an ovalbumin, a toxoid or an attenuated toxoidderivative, a cytokine, and a chemokine.

In some embodiments, the neoantigen vaccine comprises at least oneneoantigen mRNA and a pharmaceutically acceptable diluent. In someembodiments, the neoantigen vaccine comprises at least one neoantigenmRNA and a pharmaceutically acceptable adjuvant (e.g., an adjuvant asdescribed herein).

In some embodiments, the neoantigen mRNA is encapsulated by anencapsulating agent. In some embodiments, the encapsulating agentprotects the neoantigen mRNA from degradation and improves vaccinedelivery (McNamara et al. (2015) J Immunol Res. 2015:794528). In someembodiments, the encapsulating agent is a liposome. In some embodiments,the liposome is a cationic liposome such asN-[1-(2,3-dioleoloxy)propyl]-N,N,N-trimethyl ammonium chloride 1(DOTAP). In some embodiments, the encapsulating agent is a nanoparticle.In some embodiments, the nanoparticle protects the neoantigen mRNA fromnuclease degradation and/or enhances cell uptake and/or deliveryefficiency. In some embodiments, the nanoparticle may be engineered tobe fully degradable. In some embodiments, the nanoparticle is abiodegradable core-shell structured nanoparticle with a pH responsivepoly-(b-amino ester) (PBAE) core enveloped by a phospholipid shell (Suet al. (2011) Mol Pharm. 8(3):774-87). In some embodiments, suchnanoparticles are particularly efficient in delivering mRNA in vivo andeliciting an anti-tumor immune response.

In some embodiments, the subject has a non-synonymous mutational burdenof about 150 mutations or less. In some embodiments, the subject has anon-synonymous mutational burden of about 100 mutations or less. In someembodiments, the subject has a non-synonymous mutational burden of about50 mutations or less. In some embodiments, the subject has or issuspected of having a neoplastic disorder, e.g., a hematologicalmalignancy or a solid tumor. In some embodiments, the hematologicalmalignancy is selected from a B-cell malignancy, a leukemia, a lymphoma,and a myeloma. In some embodiments, the hematological malignancy isselected from acute myeloid leukemia and multiple myeloma. In someembodiments, the solid tumor is selected from breast cancer, gastriccancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer,salivary duct carcinoma, melanoma, colon cancer, cervical cancer,pancreatic cancer, kidney cancer, colorectal cancer, and esophagealcancer. In some embodiments, the solid tumor is selected fromHER2-positive breast cancer, gastric adenocarcinoma, prostate cancer,and osteosarcoma.

As used herein, “adjuvant” refers to a substance that is capable ofincreasing, amplifying, or modulating an immune response to anaccompanying immunogenic agent, e.g., a neoantigen peptide or mRNA. Incertain embodiments, a neoantigen of the present disclosure can beadministered in combination with adjuvants, i.e., substances that do notthemselves cause adaptive immune responses, but amplify or modulate theresponse to an accompanying neoantigen. A variety of adjuvants can beused in combination with the disclosed neoantigens, in order to elicitan immune response. In some embodiments, the adjuvant(s) are chosen toaugment the intrinsic response to the neoantigen without causingconformational changes in the neoantigen that would affect thequalitative form of the response. In some embodiments, the adjuvant(s)are chosen to enhance T-effector (e.g., CD8) cell priming and/oractivation.

In certain embodiments, the adjuvant is an aluminum salt (alum), such asaluminum hydroxide, aluminum phosphate, and aluminum sulphate. Suchadjuvants can be used with or without other specific immunostimulatingagents, such as 3 de-O-acylated monophosphoryl lipid A (MPL) or 3-DMP,polymeric or monomeric amino acids, such as polyglutamic acid orpolylysine. Such adjuvants can be used with or without other specificimmunostimulating agents, such as muramyl peptides (e.g.,N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP)), or other bacterial cell wall components. Otheradjuvants are oil-in-water emulsions and include (a) MF59 (WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics), (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5%pluronic-blocked polymer L121, and thr-MDP, either microfluidized into asubmicron emulsion or vortexed to generate a larger particle sizeemulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi ImmunoChem)containing 2% squalene, 0.2% Tween 80, and one or more bacterial cellwall components from the group consisting of monophosphoryllipid A(MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), forexample MPL-FCWS (Detox™). In some embodiments, the adjuvant is asaponin, such as Stimulon™ (QS21) or particles generated therefrom suchas ISCOMs (immunostimulating complexes) and ISCOMATRIX. Other adjuvantsinclude Complete Freund's Adjuvant (CFA) and Incomplete Freund'sAdjuvant (IFA), cytokines, such as interleukins (IL-1, IL-2, and IL-12),macrophage colony stimulating factor (M-CSF), and tumor necrosis factor(TNF).

An adjuvant can be administered with an immunogenic agent (e.g., aneoantigen peptide or mRNA) as a single composition, or can beadministered before, concurrent with, or after administration of theimmunogenic agent. In some embodiments, the immunogenic agent andadjuvant can be packaged and supplied in the same vial or can bepackaged in separate vials and mixed before use. In some embodiments,the immunogenic agent and adjuvant can be packaged with a label,indicating the intended therapeutic application. In some embodiments, ifthe immunogenic agent and adjuvant are packaged separately, thepackaging can include instructions for mixing before use. The choice ofan adjuvant and/or carrier depends on the stability of the immunogenicformulation containing the adjuvant, the route of administration, thedosing schedule, the efficacy of the adjuvant for the species beingvaccinated, and, in humans, a pharmaceutically acceptable adjuvant isone that has been approved or is approvable for human administration bypertinent regulatory bodies. For example, Complete Freund's adjuvant isnot suitable for human administration. However, alum, MPL or IncompleteFreund's adjuvant (Chang et al. (1998) Adv Drug Deliv Rev. 32:173-186)alone or optionally in combination with any of alum, QS21, and MPL andall combinations thereof are suitable for human administration.

In various embodiments, the present disclosure further provides methodsof screening for and identifying at least one neoantigen. Morespecifically, in various embodiments, the present disclosure provides amethod of identifying at least one neoantigen by (a) contacting aneoplastic cell with an effective amount of a splicing modulator, anADC, or a composition comprising a splicing modulator or ADC; (b)detecting at least one alternatively-spliced mRNA transcript aftercontacting the neoplastic cell with the splicing modulator, ADC, orcomposition; (c) predicting translation of the at least onealternatively-spliced mRNA transcript into at least one peptide; and (d)comparing the at least one peptide to a reference proteome, wherein atleast one neoantigen is identified if the at least one peptide does notmatch any peptides in the reference proteome. In various embodiments,the method further comprises contacting one or more additionalneoplastic cells to identify at least one universal neoantigen. Invarious embodiments, the method is repeated on one or more additionalneoplastic cells or samples (e.g., a tissue biopsy) to confirm suitableneoantigens (e.g., for use in a neoantigen vaccine) and/or to identifyone or more universal neoantigens.

In various other embodiments, the present disclosure provides a methodof identifying at least one neoantigen by (a) contacting a neoplasticcell with an effective amount of a splicing modulator, an ADC, or acomposition comprising a splicing modulator or ADC; (b) detecting atleast one peptide comprising a potential neoantigen sequence aftercontacting the neoplastic cell with the splicing modulator, ADC, orcomposition; and (c) comparing the at least one peptide to a referenceproteome, wherein at least one neoantigen is identified if the at leastone peptide does not match any peptides in the reference proteome. Invarious embodiments, the method further comprises contacting one or moreadditional neoplastic cells to identify at least one universalneoantigen. In various embodiments, the method is repeated on one ormore additional neoplastic cells or samples (e.g., a tissue biopsy) toconfirm suitable neoantigens (e.g., for use in a neoantigen vaccine)and/or to identify one or more universal neoantigens.

In some embodiments of the neoantigen identification methods describedherein, detecting at least one alternatively-spliced mRNA transcriptcomprises RNAseq. In some embodiments, predicting translation of the atleast one alternatively-spliced mRNA transcript comprises quantifyingthe change in percent spliced in (dPSI) value for the at least onetranscript. In some embodiments, predicting translation of the at leastone alternatively-spliced mRNA transcript comprises RiboSeq and/orribosomal profiling.

In some embodiments of the neoantigen identification methods describedherein, the methods further comprise evaluating the at least one peptidefor predicted major histocompatibility complex (MHC) binding. In someembodiments, predicted MHC binding is determined by measuring rawaffinity predicted binding strength of the at least one peptide. In someembodiments, a raw affinity predicted binding strength of about 500 nMor higher indicates MHC binding. In some embodiments, predicted MHCbinding is determined by identifying a distribution of predicted bindingstrengths for a series of random peptides; and comparing predictedbinding strength of the at least one peptide to the distribution. Insome embodiments, a predicted binding strength in the top 2.0% of thedistribution indicates weak MHC binding. In some embodiments, apredicted binding strength in the top 0.5% of the distribution indicatesstrong MHC binding.

In some embodiments of the neoantigen identification methods describedherein, the neoplastic cell is present in an in vitro cell culture. Insome embodiments, the neoplastic cell is obtained from the subject. Insome embodiments, the neoplastic cell is present in the subject.

Also provided herein, in various embodiments, are methods of making aneoantigen vaccine by (a) identifying at least one neoantigen (e.g., atleast one neoantigen peptide or its encoding mRNA) using any of theexemplary identification methods disclosed herein; and (b) formulatingthe at least one neoantigen together with a pharmaceutically acceptablecarrier, diluent, or adjuvant (e.g., any of the pharmaceuticallyacceptable carriers, diluents, or adjuvants described herein). In someembodiments, the at least one neoantigen used in the vaccine comprisesan amino acid sequence of any one of SEQ ID NOs: 37-65. In someembodiments, the at least one neoantigen used in the vaccine comprisesan amino acid sequence of SEQ ID NO: 37.

In some embodiments, the at least one neoantigen used in the vaccinecomprises an amino acid sequence of SEQ ID NO: 39. In some embodiments,the at least one neoantigen used in the vaccine comprises an amino acidsequence of any one of SEQ ID NOs: 46-49.

In some other embodiments, the at least one neoantigen used in thevaccine comprises an amino acid sequence of any one of SEQ ID NOs:66-93, or an antigenic portion of any one of SEQ ID NOs: 66-93. In someembodiments, the at least one neoantigen used in the vaccine comprisesan amino acid sequence of SEQ ID NO: 66, or an antigenic portion of SEQID NO: 66. In some embodiments, the at least one neoantigen used in thevaccine comprises an amino acid sequence of any one of SEQ ID NOs:74-77, or an antigenic portion of any one of SEQ ID NOs: 74-77. In someembodiments, the at least one neoantigen and/or antigenic portion rangesfrom about 10 to about 50 amino acids in length. In some embodiments,the at least one neoantigen and/or antigenic portion ranges from about10 to about 35 amino acids in length. In some embodiments, the at leastone neoantigen and/or antigenic portion ranges from about 15 to about 25amino acids in length. In some embodiments, the at least one neoantigenand/or antigenic portion ranges from about 10 to about 20 amino acids inlength. In some embodiments, the at least one neoantigen and/orantigenic portion does not exclusively overlap or consist of thecanonical peptide sequence (e.g., any of the exemplary canonical peptidesequences underlined in Table 21).

In some embodiments, the at least one neoantigen used in the vaccine islinked to the pharmaceutically acceptable carrier. In some embodiments,the pharmaceutically acceptable carrier is selected from a peptide, aserum albumin, a keyhole limpet hemocyanin, an immunoglobulin, athyroglobulin, an ovalbumin, a toxoid or an attenuated toxoidderivative, a cytokine, and a chemokine.

Combination of Splicing Modulator/ADC and Engineered T-Cells (CAR-T):

In various embodiments, a patient having a cancer as described hereincan be treated with a combination of a splicing modulator, ADC, orcomposition and one or more engineered tumor-targeting T-cells (i.e.,CAR-T). Thus, in various embodiments, the present disclosure provides amethod of treating a subject having or suspected of having a neoplasticdisorder by administering to the subject an effective amount of asplicing modulator, an ADC, or a composition comprising a splicingmodulator or ADC; and engineered tumor-targeting T-cells (i.e., CAR-T).In various embodiments, a chimeric T-cell receptor can be engineeredusing antigen recognition sequences that are reactive with an identifiedneoantigen.

For instance, in various embodiments, in order to target splicingmodulator- or ADC-induced changes in the extracellular domains of cellsurface proteins, a chimeric antigen-reactive T-cell receptor (CAR) maybe engineered by first identifying antibodies that recognize a cellsurface-expressed neoantigen protein domain. The antigen recognitionsequences of such antibodies can then be fused to a T-cell receptordomain for selective targeting and activation.

In various other embodiments, a strategy integrating the antigenpresentation machinery of tumor cells together with splicing modulator-or ADC-derived neoantigens is employed. In some embodiments, cellscontaining known and frequently represented HLA alleles (e.g.,HLA-A*02:01) can be treated with a splicing modulator, ADC, orcomposition and MHC1-bound neoantigens are identified by ligandomics. Insome embodiments, these peptides can be used to prime and/or expandT-cells from healthy donors expressing the same HLA allele. SuchT-cells, in some embodiments, can be isolated and the T-cell receptor(TCR) α and β chains sequenced to identify the cognate antigenrecognition/variable regions. In some embodiments, a cognate CAR canthen be engineered.

In some embodiments, the CAR sequences are cloned into patient-derivedT-cell populations and expanded using currently available protocols. Insome embodiments, the engineered T-cells are then transfused back intothe patient's circulation, following treatment with a splicingmodulator, ADC, or composition. After treatment with the splicingmodulator, ADC, or composition, in some embodiments, the tumor cells maybegin to present antigen. In some embodiments, the engineered T-cellpopulation can engage with and kill antigen presenting tumor cells.

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the disclosuredescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the disclosure or the embodimentsdisclosed herein. Having now described the disclosure in detail, thesame will be more clearly understood by reference to the followingexamples, which are included for purposes of illustration only and arenot intended to be limiting.

EXAMPLES Example 1

Synthesis methods for payloads, linkers, and conjugatable linker-payload(linker-drug, L-D) compounds, having the structures shown in Tables 7-9,are described.

Conjugatable linker-payloads were used in the preparation ofantibody-drug conjugates (ADCs). Exemplary ADCs are described inExamples 3-5.

1.1 Reagents and Materials

The starting materials used in the following synthesis methods areeither commercially available or can be readily prepared by standardmethods from known materials. The disclosed conjugatable linker-payloadscan be prepared using the reactions and techniques described herein. Inthe description of the synthetic methods described below, it is to beunderstood that all proposed reaction conditions, including choice ofsolvent, reaction atmosphere, reaction temperature, duration of theexperiment, and workup procedures, can be chosen to be the conditionsstandard for that reaction, unless otherwise indicated. It is understoodby one skilled in the art of organic synthesis that the functionalitypresent on various portions of the molecule should be compatible withthe reagents and reactions proposed. Substituents not compatible withthe reaction conditions are apparent to one skilled in the art, andalternate methods are therefore indicated herein.

Preparative liquid chromatography-mass spectrometry (LC/MS) wasconducted using a Waters AutoPurification System and an XTerra MS C18column (5 μm, 19 mm×100 mm) under acidic mobile phase conditions.Nuclear magnetic resonance (NMR) spectra were recorded at 400 MHz usinga Varian instrument (Agilent Technologies). Microwave heating wasperformed using a Biotage Emrys Liberator or Initiator microwave. Columnchromatography was carried out using a Teledyne Isco Combiflash Rf200d.Solvent removal was carried out using either a Büchi rotary evaporatoror a Genevac centrifugal evaporator.

Terms/Abbreviations: As used herein, the term “inerted” refers toreplacement of the air in a reactor (e.g., a reaction vessel, a flask, aglass reactor) with an essentially moisture-free, inert gas, such asnitrogen or argon. The following abbreviations are used herein:DCM=dichloromethane, DMF=dimethylformamide, HPLC=high performance liquidchromatography, KHMDS=potassium bis(trimethylsilyl)amide, LC/MS=liquidchromatography-mass spectrometry, MeOH=methanol, RT=room temperature,TBSCl=tert-butyldimethylsilyl chloride, THF=tetrahydrofuran,TLC=thin-layer chromatography. Multiplicities are indicated using thefollowing abbreviations: s=singlet, d=doublet, t=triplet, q=quartet,quint=quintet, sxt=sextet, m=multiplet, dd=doublet of doublets,ddd=doublet of doublets of doublets, dt=doublet of triplets, br s=abroad singlet.

LC/MS: Mobile phases=A (0.1% formic acid in H2O) and B (0.1% formic acidin acetonitrile). Gradient=B 5% to 95% in 1.8 min. Column=Waters AcquityBEH C18 column (1.7 μm, 2.1×50 mm).

References: U.S. Pat. Nos. 7,884,128 and 7,816,401 describe exemplarymethods of synthesizing pladienolide B and D and are each incorporatedherein by reference for such methods. Synthesis of pladienolide B and Dmay also be performed using the exemplary methods described in Kanada etal. ((2007) Angew Chem Int Ed. 46:4350-5). Kanada et al. and Intl. Pub.No. WO 2003/099813 describe exemplary methods for synthesizing E7107(D11) (Compound 45 of WO 2003/099813) from Pladienolide D (11107D of WO2003/099813). A corresponding U.S. Pat. No. is U.S. Pat. No. 7,550,503to Kotake et al. Each of these references is incorporated herein for thedescribed synthesis methods.

TABLE 7 Structures of exemplary drug moieties (payloads) PayloadStructure/ID (Payload Series)

TABLE 8 Structures of exemplary linkers Linker Structure/ID (IUPAC Name)

TABLE 9 Structures of exemplary conjugatable linker-payload (L-D)compounds

1.2 Preparation of Pladienolide-Based Payloads 1.2.1 Overview—GeneralProcedure 1

Step 1:(2S,3S,6S,7R,10R,E)-7,10-dihydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylacetate (1.7 g, 3.154 mmol), triethylamine (4.40 mL, 31.536 mmol),1,2-dichloroethane (31.5 mL, 3.154 mmol) were combined and stirred atRT. Chlorotriethylsilane (2.1 mL, 12.615 mmol) was added and stirredovernight. Brine was poured into the reaction mix and stirred for 30 minand the organic layer separated. The aqueous layer was back extractedwith DCM (3×). The organic layers were combined, dried (an. Na₂SO₄),concentrated to dryness, and chromatographed to afford(2S,3S,6S,7R,10R,E)-7-hydroxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylacetate (1.274 g, 1.423 mmol, 45.1% yield).

¹H-NMR (400 MHz, DMSO-d6): δ ppm 0.51-0.64 (m, 18H) 0.74-0.84 (m, 9H)0.90-0.97 (m, 26H) 1.04 (s, 3H) 1.18 (s, 4H) 1.37 (s, 3H) 1.41-1.57 (m,4H) 1.69 (s, 3H) 1.84-1.93 (m, 1H) 2.00-2.05 (m, 3H) 2.24-2.35 (m, 1H)2.38-2.45 (m, 1H) 2.72-2.80 (m, 1H) 3.28-3.30 (m, 1H) 3.62-3.70 (m, 1H)3.80-3.90 (m, 1H) 4.56 (s, 1H) 4.81-4.93 (m, 2H) 5.41-5.52 (m, 1H)5.63-5.73 (m, 1H) 5.77-5.87 (m, 1H) 6.01-6.12 (m, 1H) 6.42 (dd, J=15.12,11.11 Hz, 1H).

Step 2: To a solution of(2S,3S,6S,7R,10R,E)-7-hydroxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylacetate (0.7 g, 0.782 mmol) in toluene (4.16 mL, 39.085 mmol) was added1,8-Naphthalenediamine, N,N,N′,N′-tetramethyl- (1.173 g, 5.472 mmol),followed by methyl trifluoromethanesulfonate (0.354 mL, 3.127 mmol) at0° C. The reaction mixture was then warmed up to 50° C. and heated for 3hours. Solvent was evaporated. Purification by column chromatographyafforded(2S,3S,6S,7R,10R,E)-7-methoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylacetate (467 mg, 0.513 mmol, 65.7% yield) as a colorless oil.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.62-0.70 (m, 18H) 0.83-0.95 (m, 13H)0.99-1.07 (m, 27H) 1.22 (s, 3H) 1.24-1.36 (m, 2H), 1.28-1.28 (m, 1H)1.45 (s, 3H) 1.47-1.65 (m, 8H) 1.78 (d, J=0.88 Hz, 3H) 1.91-2.00 (m, 1H)2.07 (s, 3H) 2.37-2.45 (m, 1H) 2.51-2.68, (m, 3H) 2.88-2.92 (m, 1H)3.20-3.24 (m, 1H) 3.35 (s, 3H) 3.74-3.82 (m, 1H) 3.93-4.02 (m, 1H)4.94-4.99 (m, 1H) 5.08-5.14 (m, 1H), 5.53-5.64 (m, 1H) 5.70-5.79 (m, 1H)5.81-5.88 (m, 1H) 6.12-6.18 (m, 1H) 6.47-6.58 (m, 1H).

Step 3:(2S,3S,6S,7R,10R,E)-7-hydroxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylacetate (500 mg, 0.558 mmol) was dissolved in MeOH (6009 μL, 148.524mmol) and potassium carbonate (232 mg, 1.675 mmol) was added. Thereaction mixture was stirred at RT for 1 hour and showed the reactioncomplete. The reaction mixture was diluted with water and extracted withEtOAc (3×). The combined organics were washed with brine, dried oversodium sulfate, filtered, and concentrated. Purification by columnchromatography afforded(4R,7R,8S,11S,12S,E)-7,8-dihydroxy-7,11-dimethyl-12-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-4-((triethylsilyl)oxy)oxacyclododec-9-en-2-one(305 mg, 0.357 mmol, 64.0% yield).

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.61-0.72 (m, 18H) 0.82-0.92 (m, 7H)0.92-1.06 (m, 30H) 1.19-1.40 (m, 7H) 1.42-1.66 (m, 8H) 1.51-1.52 (m, 1H)1.75-1.81 (m, 3H) 1.91-2.01 (m, 1H) 2.34-2.45 (m, 1H) 2.51-2.61 (m, 2H)2.61-2.69 (m, 1H) 2.86-2.94 (m, 1H) 3.67-3.73 (m, 1H) 3.73-3.80 (m, 1H)3.89-3.96 (m, 1H) 4.08-4.17 (m, 1H) 4.93-4.99 (m, 1H) 5.36-5.47 (m, 1H)5.67-5.78 (m, 1H) 5.80-5.88 (m, 1H) 6.10-6.19 (m, 1H) 6.47-6.58 (m, 1H).

Step 4:(4R,7R,8S,11S,12S,E)-8-hydroxy-7-methoxy-7,11-dimethyl-12-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-4-((triethylsilyl)oxy)oxacyclododec-9-en-2-one(0.386 g, 0.445 mmol), DCM (0.1 M), DMAP (1.0 equiv.), Hunig's base (5.0equiv.), 4-Nitrophenyl chloroformate (1.8 equiv.) were combined andstirred overnight. The reaction mixture was then extracted with 1N NaOH.The organic layer was dried (an. Na₂SO₄) and concentrated to dryness.The residue was mixed with DCM (0.1 M), Hunig's base (5.0 equiv.), amine(2.0 equiv.), which were combined and stirred for 1 hour. The reactionmixture was concentrated and chromatographed to afford thetriTES-Pladienolide carbamate.

Step 5: triTES-Pladienolide carbamate (1 equiv.), DCM (0.04 M), andDIPEA (191 equiv.) were combined and cooled to −78° C. Hydrogenfluoride-pyridine (6.2 equiv.) was added and the reaction was allowed towarm to RT and stirred overnight. The reaction mixture was cooled in anice bath, then sat. NaHCO₃ was added, stirred, and extracted with DCM.The organic layers were combined, dried over an. Na₂SO₄, concentrated,and chromatographed to afford the pladienolide carbamate.

1.2.1.1 D1

General procedure 1 (outlined in section 1.2.1) was employed tosynthesize D1.

(2S,3S,6S,7R,10R,E)-7-methoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylpiperazine-1-carboxylate (240 mg, 0.245 mmol, 55.1% yield). LC/MS (ESI,m/z), 980.4 [M+H]⁺.

¹H-NMR (400 MHz, CDCl₃-d): δ ppm 0.56-0.66 (m, 18H) 0.78-0.91 (m, 9H)0.96 (t, J=7.91 Hz, 27H) 1.18-1.22 (m, 3H) 1.23-1.28 (m, 1H) 1.37-1.41(m, 3H) 1.41-1.63 (m, 7H) 1.69-1.74 (m, 3H) 1.89 (dd, J=13.87, 4.96 Hz,1H) 2.33-2.62 (m, 4H) 2.78-2.89 (m, 5H), 3.35 (s, 3H) 3.43-3.52 (m, 4H)3.73 (td, J=6.40, 3.51 Hz, 1H) 3.86 (br dd, J=7.84, 3.95 Hz, 1H)4.94-5.13 (m, 2H) 5.58-5.76 (m, 3H) 6.12, (br d, J=0.75 Hz, 1H) 6.41(dd, J=15.06, 11.04 Hz, 1H).

D1

(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.83-1.01 (m, 10H) 1.23 (s, 3H)1.25-1.32 (m, 1H) 1.35 (s, 3H) 1.40-1.61 (m, 6H) 1.61-1.71 (m, 2H) 1.79(d, J=0.75 Hz, 3H) 1.84-1.93 (m, 1H) 2.45-2.63 (m, 3H) 2.63-2.72 (m, 1H)2.77-2.85 (m, 4H) 2.86-2.94 (m, 1H) 3.33-3.37 (m, 3H) 3.40-3.57 (m, 5H)3.77-3.89 (m, 1H) 4.38-4.42 (m, 1H) 5.01-5.12 (m, 2H) 5.52-5.65 (m, 1H)5.69-5.80 (m, 1H) 5.84-5.92 (m, 1H) 6.09-6.18 (m, 1H) 6.49-6.60 (m, 1H).

1.2.1.2 D2

General procedure 1 (outlined in section 1.2.1) was employed tosynthesize D2.

(2S,3S,6S,7R,10R,E)-7-methoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-yl4-methylpiperazine-1-carboxylate (413 mg, 0.416 mmol, 90% yield). LC/MS(ESI, m/z), 994 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.61-0.71 (m, 18H) 0.83-0.94 (m, 9H)0.98-1.07 (m, 28H) 1.23 (s, 3H) 1.41-1.48 (m, 3H) 1.48-1.65 (m, 7H)1.72-1.81 (m, 3H) 1.93-1.99 (m, 1H) 2.03 (s, 3H) 2.32 (s, 3H) 2.38-2.48(m, 5H) 2.52-2.67 (m, 3H) 2.87-2.93 (m, 1H) 3.47-3.61 (m, 4H) 3.72-3.81(m, 1H) 3.96-4.04 (m, 1H) 4.53-4.62 (m, 1H) 4.93-5.07 (m, 2H) 5.51-5.64(m, 1H) 5.69-5.79 (m, 1H) 5.81-5.92 (m, 1H) 6.09-6.21 (m, 1H) 6.46-6.60(m, 1H).

D2

(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-methylpiperazine-1-carboxylate (37.8 mg, 0.058 mmol, 24.05% yield).LC/MS (ESI, m/z), 651.69 [M+H]⁺.

¹H-NMR (400 MHz, DMSO-d6): δ ppm 0.76-0.89 (m, 9H) 1.10 (s, 3H)1.20-1.26 (m, 3H) 1.30-1.42 (m, 4H) 1.42-1.55 (m, 2H) 1.66-1.75 (m, 3H)1.75-1.82 (m, 1H) 2.13-2.21 (m, 3H) 2.25 (br s, 4H) 2.31-2.41 (m, 2H)2.54-2.60 (m, 2H) 2.72-2.82 (m, 1H) 3.22 (s, 3H) 3.36-3.40 (m, 3H)3.66-3.76 (m, 1H) 4.36-4.46 (m, 1H) 4.53-4.60 (m, 1H) 4.78-4.85 (m, 1H)4.86-4.96 (m, 2H) 5.36-5.50 (m, 1H) 5.60-5.74 (m, 1H) 5.80-5.92 (m, 1H)6.02-6.11 (m, 1H) 6.34-6.46 (m, 1H).

1.3 Preparation of MC-Val-Cit-pABC Linker-Payloads 1.3.1Overview—General Procedure 1

Payload (1.0 equiv.), Hunig's base (3.0 equiv.), DMF (0.1 M), and4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (1.2 equiv.) were combined and stirred at RTovernight. The reaction mixture was then concentrated and purified viacolumn chromatography (MeOH in DCM) or reverse-phase HPLC to afford theproduct.

1.3.1.1 ADL1-D1

Linker-Payload (ADL1-D1): General procedure 1 (outlined in section1.3.1) was employed to synthesize1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)4-((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazine-1,4-dicarboxylate (50 mg, 0.040 mmol, 42 yield). LC/MS (ESI,m/z), 1258.5 [M+Na]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.85-1.04 (m, 16H) 1.18-1.26 (m, 3H)1.26-1.38 (m, 6H) 1.40-1.71 (m, 14H) 1.80 (s, 3H) 1.85-1.98 (m, 2H)2.02-2.15 (m, 1H) 2.24-2.34 (m, 2H) 2.44-2.64 (m, 3H) 2.65-2.72 (m, 1H)2.87-2.96 (m, 1H) 3.06-3.27 (m, 2H) 3.37 (s, 6H) 3.43-3.61 (m, 12H)3.79-3.90 (m, 1H) 4.12-4.21 (m, 1H) 4.48-4.55 (m, 1H) 5.02-5.14 (m, 4H)5.55-5.65 (m, 1H), 5.69-5.81 (m, 1H) 5.85-5.93 (m, 1H) 6.12-6.19 (m, 1H)6.49-6.60 (m, 1H) 6.81 (s, 2H) 7.29-7.38 (m, 2H) 7.57-7.65 (m, 2H).

1.3.1.2 ADL1-D18

The payload D18 was prepared using procedures outlined in section 1.2.1,employing pladienolide B as the starting material (SM) (R═H; Scheme 1).

(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((S,2E,4E)-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (205 mg, 0.330 mmol, 77% yield). LC/MS (ESI,m/z), 621.6 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.88-0.99 (m, 9H) 1.10 (d, J=6.78 Hz,3H) 1.24 (s, 4H) 1.42-1.69 (m, 8H) 1.77 (d, J=0.88 Hz, 3H) 2.43-2.63 (m,4H) 2.64-2.70 (m, 1H) 2.71-2.82 (m, 5H) 3.34 (br s, 3H) 3.37 (s, 2H)3.42-3.57 (m, 5H) 3.79-3.89 (m, 1H) 5.06 (s, 2H), 5.54-5.63 (m, 1H)5.64-5.80 (m, 2H) 6.07-6.16 (m, 1H) 6.29-6.40 (m, 1H).

Linker-Payload (ADL1-D18): General procedure 1 (outlined in section1.3.1) was employed to synthesize1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)4-((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((S,2E,4E)-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazine-1,4-dicarboxylate (72 mg, 0.059 mmol, 54.7% yield). LC/MS(ESI, m/z), 1220.08 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.87-1.02 (m, 15H) 1.10 (d, J=6.78 Hz,3H) 1.23 (s, 4H) 1.29-1.38 (m, 2H) 1.44-1.68 (m, 13H), 1.54-1.55 (m, 1H)1.77 (d, J=0.75 Hz, 4H) 1.87-1.95 (m, 1H) 2.05-2.15 (m, 1H) 2.24-2.32(m, 2H) 2.45-2.62 (m, 4H) 2.65-2.70 (m, 1H), 2.71-2.78 (m, 1H) 3.13-3.18(m, 2H) 3.46-3.58 (m, 11H) 3.79-3.90 (m, 1H) 4.13-4.21 (m, 1H) 4.47-4.56(m, 1H) 5.11 (s, 4H) 5.53-5.81 (m, 3H) 6.06-6.16 (m, 1H) 6.29-6.40 (m,1H) 6.81 (s, 2H) 7.34 (d, J=8.53 Hz, 2H) 7.60 (d, J=8.53 Hz, 2H).

1.3.1.3 ADL1-D8 & ADL6-D8

The payload D8 was synthesized according to the procedure outlinedbelow:

Step 1: To a solution of tri-TES-Pladienolide D (200 mg, 0.223 mmol) indichloromethane (2 mL) at 0° C. was added DMAP (409 mg, 3.35 mmol) and4-nitrophenyl chloroformate (338 mg, 1.675 mmol). The reaction mixturewas stirred at RT for 7 days, diluted with EtOAc and water, then thelayers were separated. The aqueous layer was extracted with EtOAc (2×),and the combined organic extracts were washed with brine. The combinedorganic layers were dried over Na₂SO₄, filtered and concentrated invacuo. Flash chromatography afforded(2S,3S,6S,7R,10R,E)-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-7-(((4-nitrophenoxy)carbonyl)oxy)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylacetate. (170 mg, 72% yield).

¹H-NMR (400 MHz, CHCl₃-d): δ ppm 0.54-0.67 (m, 18H) 0.78-1.03 (m, 36H)1.19-1.32 (m, 1H) 1.39 (s, 3H) 1.43-1.52 (m, 3H) 1.55-1.63 (m, 3H) 1.64(s, 3H) 1.74 (s, 3H) 1.88 (dd, J=13.80, 5.02 Hz, 1H) 2.13 (s, 3H)2.23-2.37 (m, 1H) 2.39-2.48 (m, 2H) 2.51-2.63 (m, 2H) 2.84 (s, 1H)3.69-3.77 (m, 1H) 3.82-4.00 (m, 1H) 5.04 (d, J=10.79 Hz, 1H) 5.24 (d,J=9.03 Hz, 1H) 5.67-5.84 (m, 3H) 6.12 (d, J=10.16 Hz, 1H) 6.42 (dd,J=15.06, 11.04 Hz, 1H) 7.42 (d, J=9.29 Hz, 2H) 8.29 (d, J=9.16 Hz, 2H).

Step 2: To a solution of(2S,3S,6S,7R,10R,E)-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-7-(((4-nitrophenoxy)carbonyl)oxy)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylacetate (100 mg, 0.094 mmol) in DCM was added piperazine and DMAP. Theresulting yellowish suspension was stirred for 6 hours. The reactionmixture was concentrated to give the crude product. Flash chromatographyafforded(2S,3S,6S,7R,10R,E)-6-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-7-ylpiperazine-1-carboxylate (95 mg, 100%). LC/MS (ESI, m/z), 1008.8 [M+H]⁺.

¹H-NMR (400 MHz, CHCl₃-d): δ ppm 0.42-0.70 (m, 22H) 0.79-0.84 (m, 7H)0.86-0.91 (m, 4H) 0.92-1.03 (m, 30H) 1.15-1.30 (m, 2H) 1.37-1.42 (m, 3H)1.44-1.52 (m, 3H) 1.56-1.62 (m, 2H) 1.62-1.68 (m, 1H) 1.71-1.76 (m, 3H)1.83-1.93 (m, 1H) 2.03-2.11 (m, 4H) 2.36-2.45 (m, 2H) 2.45-2.53 (m, 2H)2.54-2.64 (m, 1H) 2.78-2.86 (m, 1H) 2.86-3.07 (m, 4H) 3.32-3.45 (m, 1H)3.45-3.64 (m, 3H) 3.69-3.78 (m, 1H) 3.79-3.94 (m, 1H) 5.00 (d, J=10.54Hz, 1H) 5.18 (s, 1H) 5.54-5.79 (m, 3H) 5.98-6.21 (m, 1H) 6.33-6.57 (m,1H) 6.84-6.96 (m, 3H) 8.02-8.35 (m, 2H) 8.06-8.08 (m, 1H).

Step 3: To a solution of(2S,3S,6S,7R,10R,E)-6-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-7-ylpiperazine-1-carboxylatein (95 mg, 0.094 mmol) in THF (3 mL) was added TBAF (0.424 mL, 1 M,0.424 mmol) and stirred at RT for 10 hours. The mixture as concentratedand diluted with EtOAc, washed with water and brine. The organic layerwas separated and dried with Na₂SO₄, filtered and concentrated in vacuo.HPLC purification afforded(2S,3S,6S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-7-ylpiperazine-1-carboxylate (16 mg, 26%). LC/MS (ESI, m/z), 665.6 [M+H]⁺.

¹H-NMR (400 MHz, CHCl₃-d): δ ppm 0.90 (dd, J=6.84, 2.20 Hz, 6H) 0.94 (t,J=7.40 Hz, 3H) 1.20-1.30 (m, 1H) 1.34 (s, 3H) 1.39-1.54 (m, 3H) 1.55 (s,3H) 1.59-1.73 (m, 3H) 1.78 (d, J=0.88 Hz, 3H) 1.86 (dd, J=13.99, 5.46Hz, 1H) 2.05 (s, 3H) 2.39-2.53 (m, 3H) 2.55-2.65 (m, 1H) 2.67 (dd,J=8.03, 2.26 Hz, 1H) 2.89 (s, 1H) 3.22 (br s, 4H) 3.50-3.57 (m, 1H)3.58-3.90 (m, 5H) 5.08 (d, J=10.67 Hz, 1H) 5.18 (d, J=9.03 Hz, 1H)5.58-5.78 (m, 2H) 5.88 (d, J=15.31 Hz, 1H) 6.10-6.23 (m, 1H) 6.53 (dd,J=15.25, 10.98 Hz, 1H).

Step 4:

Linker-Payload (ADL1-D8):4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (7.5 mg, 10.166 μmol) in DMF (315 μL, 4.066mmol) was added Hunig's base (5.33 μL, 0.03 mmol). The reaction wascooled to 0° C., and added(2S,3S,6S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-7-ylpiperazine-1-carboxylate(7.50 mg, 0.011 mmol). The reaction mixture was stirred at RT untilconsumption of starting material. The reaction mixture was concentratedin vacuo. Flash chromatography afforded ADL1-D8 (7.2 mg, 5.70 μmol,56.1% yield). LC/MS (ESI, m/z), 1263.8 [M+H]⁺. General procedure 1(1.3.1) can also be employed for the preparation of ADL1-110987.

¹H-NMR (400 MHz, CHLOROFORM-d): δ ppm 0.80-1.02 (m, 16H) 1.20-1.35 (m,6H) 1.38-1.48 (m, 2H) 1.57-1.69 (m, 7H) 1.74-1.81 (m, 4H) 1.86-1.96 (m,2H) 2.00-2.11 (m, 4H) 2.31 (br d, J=6.02 Hz, 2H) 2.45-2.55 (m, 2H)2.59-2.74 (m, 2H) 2.85-2.96 (m, 1H) 3.05-3.25 (m, 5H) 3.40-3.59 (m, 9H)3.62-3.88 (m, 1H) 4.15 (d, J=7.53 Hz, 1H) 5.05-5.22 (m, 5H) 5.56-5.77(m, 2H) 5.84-5.98 (m, 1H) 6.11-6.22 (m, 1H) 6.47-6.61 (m, 1H) 7.31-7.40(m, 2H) 7.56-7.65 (m, 2H).

Linker-Payload (ADL6-D8):4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl) carbonate (5 mg, 7.673 μmol) in DMF (238 μL, 3.069 mmol)was added Hunig's base (4.02 μL, 0.023 mmol). The reaction mixture wascooled to 0° C., and added(2S,3S,6S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-7-ylpiperazine-1-carboxylate. The reaction mixture was stirred at RT untilconsumption of starting material. The reaction mixture was concentratedin vacuo. HPLC purification afforded ADL6-D8 (1.2 mg, 1.019 μmol, 13.28%yield). LC/MS (ESI, m/z), 1199.9 [M+Na]⁺.

¹H NMR (400 MHz, CHCl₃-d): δ ppm 0.78-1.04 (m, 18H) 1.15-1.52 (m, 58H)1.64-1.72 (m, 3H) 1.78 (d, J=6.53 Hz, 4H) 2.04-2.19 (m, 3H) 2.21-2.30(m, 1H) 2.44-2.67 (m, 4H) 2.76 (dd, J=6.09, 2.95 Hz, 1H) 2.90-3.02 (m,1H) 3.42-3.57 (m, 8H) 3.61-3.70 (m, 1H) 3.77 (br s, 1H) 4.10-4.27 (m,1H) 4.58 (br d, J=6.65 Hz, 1H) 5.01-5.26 (m, 4H) 5.52-5.70 (m, 2H) 5.88(s, 1H) 5.93-6.02 (m, 1H) 6.08-6.18 (m, 1H) 6.47-6.58 (m, 1H) 7.29-7.35(m, 2H) 7.55 (d, J=8.66 Hz, 1H).

1.3.1.4 ADL1-D4

ADL1-D4 was synthesized using the procedures outlined below.

Step 1:(2S,3S,6S,7R,10R,E)-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-6-(((4-nitrophenoxy)carbonyl)oxy)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-7-ylacetate.

To a solution of tri-TES Pladienolide D (160 mg, 0.179 mmol) in1,2-dichloroethane (5 mL) at 20° C. was added DMAP (32.7 mg, 0.268mmol), triethyl amine (0.75 mL, 5.36 mmol) and 4-nitrophenylchloroformate (360 mg, 1.787 mmol). The reaction mixture was stirred at40° C. for 4 days, and at 60° C. for 2 hours. The reaction mixture wasdiluted with EtOAc and washed with water, then the layers wereseparated. The aqueous layer was extracted with EtOAc (2×). The combinedorganic extracts were successively washed with water and brine, driedover MgSO₄, filtered, and concentrated in vacuo. Flash chromatographyafforded(2S,3S,6S,7R,10R,E)-7-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylpiperazine-1-carboxylate (150 mg, 79% yield).

¹H-NMR (400 MHz, CHCl₃-d): δ ppm 0.48-0.71 (m, 24H) 0.78-0.85 (m, 7H)0.86-0.93 (m, 5H) 0.94-1.03 (m, 34H) 1.18-1.22 (m, 2H) 1.22-1.26 (m, 2H)1.35-1.43 (m, 4H) 1.43-1.52 (m, 4H) 1.54 (s, 4H) 1.56-1.65 (m, 3H)1.68-1.72 (m, 3H) 1.75 (br d, J=0.75 Hz, 2H) 1.84-1.95 (m, 1H) 2.01-2.06(m, 2H) 2.09 (s, 2H) 2.11 (s, 2H) 2.33-2.52 (m, 4H) 2.57 (dd, J=8.09,2.07 Hz, 2H) 2.80-2.90 (m, 1H) 3.66-3.80 (m, 1H) 3.82-3.93 (m, 2H)4.92-5.13 (m, 2H) 5.63-5.68 (m, 1H) 5.69-5.74 (m, 1H) 5.75-5.83 (m, 2H)6.12 (br d, J=10.67 Hz, 1H) 6.41 (did, J=15.15, 11.01, 5.08 Hz, 1H) 7.50(d, J=9.41 Hz, 2H) 8.35 (d, J=9.29 Hz, 2H).

Step 2:(2S,3S,6S,7R,10R,E)-7-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylpiperazine-1-carboxylate.

To a solution of(2S,3S,6S,7R,10R,E)-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-6-(((4-nitrophenoxy)carbonyl)oxy)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-7-ylacetate in DCM (1 mL) was added piperazine (0.447 g, 5.195 mmol) andHunig's base (0.9 mL, 5.195 mmol). The resulting yellowish suspensionwas stirred for 6 hours. Reaction mixture was concentrated andchromatographed over silica gel to afford(2S,3S,6S,7R,10R,E)-7-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylpiperazine-1-carboxylate (1.0 g, 0.844 mmol, 81% yield). LC/MS (ESI,m/z), 1008.1 [M+H]⁺.

D4

Step 3:(2S,3S,6S,7R,10R,E)-7-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylpiperazine-1-carboxylate (1.09 g, 0.92 mmol), DCM (20.71 mL, 321.826mmol), and DIPEA (19.91 mL, 114.018 mmol) were combined and cooled to−78° C. Hydrogen fluoride-pyridine (0.518 g, 5.232 mmol) was added andthe reaction allowed to warm to RT and stirred overnight. LC/MSsuggested desilylation. The reaction mixture was cooled in an ice bath.Saturated NaHCO₃ was added and stirred and extracted with DCM. Theorganic layers were combined, dried over an. Na₂SO₄ and concentrated andchromatographed to afford(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (225 mg, 36.8%). LC/MS (ESI, m/z), 665.6[M+H]⁺.

¹H-NMR (400 MHz, CHCl₃-d): δ ppm 0.87-0.92 (m, 6H) 0.94 (t, J=7.40 Hz,3H) 1.16-1.31 (m, 1H) 1.35 (s, 3H) 1.40-1.56 (m, 4H) 1.59 (s, 3H) 1.66(br dd, J=14.68, 7.03 Hz, 3H) 1.76-1.80 (m, 3H) 1.87 (dd, J=14.12, 5.46Hz, 1H) 2.05 (s, 3H) 2.30-2.41 (m, 1H) 2.50 (d, J=3.76 Hz, 2H) 2.56-2.72(m, 2H) 2.90 (br d, J=2.01 Hz, 1H) 3.19 (br t, J=5.14 Hz, 4H) 3.50-3.59(m, 1H) 3.71 (br s, 4H) 3.77-3.89 (m, 1H) 5.01-5.13 (m, 2H) 5.58-5.71(m, 1H) 5.71-5.81 (m, 1H) 5.88 (d, J=15.31 Hz, 1H) 6.15 (br d, J=10.79Hz, 1H) 6.53 (dd, J=15.18, 10.92 Hz, 1H).

AD1-D4

To4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (23 mg, 0.031 mmol) in DMF (966 μL) in around-bottom flask was added Hunig's base (16.33 μL, 0.094 mmol). Thereaction mixture was cooled to 0° C., and D4 (22.80 mg, 0.034 mmol) wasadded and stirred at RT. The reaction mixture was concentrated in vacuo.Flash chromatography afforded ADL1-D4 (30.5 mg, 0.024 mmol, 77% yield).LC/MS (ESI, m/z), 1263.8 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d₄): δ ppm 0.87-0.93 (m, 7H) 0.93-1.01 (m, 8H)1.19-1.34 (m, 4H) 1.50 (s, 3H) 1.57 (s, 5H) 1.58-1.70 (m, 6H) 1.70-1.77(m, 1H) 1.78 (s, 3H) 1.83-1.95 (m, 2H) 2.04 (s, 3H) 2.05-2.13 (m, 1H)2.27 (t, J=7.40 Hz, 2H) 2.32-2.42 (m, 1H) 2.50 (d, J=3.64 Hz, 2H)2.55-2.74 (m, 2H) 2.90 (td, J=5.83, 2.26 Hz, 1H) 3.03-3.26 (m, 2H) 3.35(s, 13H) 3.42-3.61 (m, 11H) 3.80 (br dd, J=9.85, 3.58 Hz, 1H) 4.16 (d,J=7.40 Hz, 1H) 4.50 (dd, J=8.91, 5.14 Hz, 1H) 4.56 (s, 1H) 5.05 (dd,J=14.37, 10.10 Hz, 2H) 5.09 (s, 2H) 5.49 (s, 1H) 5.59-5.69 (m, 1H)5.72-5.80 (m, 1H) 5.87 (d, J=15.18 Hz, 1H) 6.09-6.22 (m, 1H) 6.44-6.60(m, 1H) 7.32 (d, J=8.66 Hz, 2H) 7.58 (d, J=8.53 Hz, 2H).

1.3.1.5 ADL1-D9, ADL6-D9, & ADL1-D13 D9 & D13

D9 and D13 were synthesized as a 3:1 mixture of isomers employingprocedures outlined in the synthesis of D4 (Scheme 4) utilizing tri-TESPladienolide B.

Payload (D9): LC/MS (ESI, m/z), 649.7 [M+H]⁺.

¹H-NMR (400 MHz, CHCl₃-d): δ ppm 0.87-1.01 (m, 10H) 1.08 (d, J=6.78 Hz,3H) 1.27-1.63 (m, 12H) 1.66-1.74 (m, 1H) 1.76 (s, 3H) 1.97-2.10 (m, 3H)2.35-2.57 (m, 5H) 2.58-2.65 (m, 1H) 2.65-2.71 (m, 1H) 2.77 (td, J=5.93,2.32 Hz, 1H) 2.89-3.05 (m, 4H) 3.07-3.34 (m, 8H) 3.50-3.68 (m, 5H)3.72-3.88 (m, 1H) 4.88-5.09 (m, 1H) 5.18 (d, J=10.67 Hz, 1H) 5.50-5.84(m, 3H) 6.01-6.13 (m, 1H) 6.19-6.36 (m, 1H).

Payload (D13): LC/MS (ESI, m/z), 649.6 [M+H]⁺.

¹H NMR (400 MHz, CHCl₃-d) δ ppm 0.84-1.01 (m, 11H) 1.08 (d, J=6.78 Hz,3H) 1.29-1.64 (m, 10H) 1.63-1.73 (m, 1H) 1.76 (s, 3H) 1.94-2.12 (m, 4H)2.37-2.58 (m, 4H) 2.59-2.65 (m, 1H) 2.68 (dd, J=7.40, 2.26 Hz, 1H) 2.77(td, J=5.93, 2.32 Hz, 1H) 3.01-3.30 (m, 4H) 3.49 (s, 1H) 3.54-3.67 (m,2H) 3.69-3.92 (m, 5H) 4.13-4.78 (m, 11H) 5.13-5.24 (m, 2H) 5.48-5.61 (m,1H) 5.62-5.74 (m, 2H) 6.04-6.13 (m, 1H) 6.18-6.32 (m, 1H).

ADL1-D9

General procedure 1 (outlined in section 1.3.1) was employed tosynthesize

ADL1-D9.

Linker-Payload (ADL1-D9): (30.5 mg, 0.024 mmol, 77% yield). LC/MS (ESI,m/z), 1263.8 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d₄): δ ppm 0.87-0.93 (m, 7H) 0.93-1.01 (m, 8H)1.19-1.34 (m, 4H) 1.50 (s, 3H) 1.57 (s, 5H) 1.58-1.70 (m, 6H) 1.70-1.77(m, 1H) 1.78 (s, 3H) 1.83-1.95 (m, 2H) 2.04 (s, 3H) 2.05-2.13 (m, 1H)2.27 (t, J=7.40 Hz, 2H) 2.32-2.42 (m, 1H) 2.50 (d, J=3.64 Hz, 2H)2.55-2.74 (m, 2H) 2.90 (td, J=5.83, 2.26 Hz, 1H) 3.03-3.26 (m, 2H) 3.35(s, 13H) 3.42-3.61 (m, 11H) 3.80 (br dd, J=9.85, 3.58 Hz, 1H) 4.16 (d,J=7.40 Hz, 1H) 4.50 (dd, J=8.91, 5.14 Hz, 1H) 4.56 (s, 1H) 5.05 (dd,J=14.37, 10.10 Hz, 2H) 5.09 (s, 2H) 5.49 (s, 1H) 5.59-5.69 (m, 1H)5.72-5.80 (m, 1H) 5.87 (d, J=15.18 Hz, 1H) 6.09-6.22 (m, 1H) 6.44-6.60(m, 1H) 7.32 (d, J=8.66 Hz, 2H) 7.58 (d, J=8.53 Hz, 2H).

ADL6-D9

General procedure 1 (outlined in section 1.3.1) was employed tosynthesize ADL6-D9.

Linker-Payload (ADL6-D9): To diluted4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl) carbonate (16.5 mg, 0.025 mmol) in DMF (784 μL) wasadded Hunig's Base (13.27 μL, 0.076 mmol). The reaction mixture wascooled to 0° C., and D9 was added. The reaction mixture was stirred atRT until LC/MS showed the reaction complete. The reaction mixture wasconcentrated in vacuo. Flash chromatography of the residue on silica gelwith DCM/MeOH gave the titled compound (16.7 mg, 57% yield). LC/MS (ESI,m/z), 1184.6 [M+Na]⁺.

¹H-NMR (400 MHz, MeOH-d₄): δ ppm 0.80-1.02 (m, 16H) 1.05-1.12 (m, 4H)1.13-1.34 (m, 5H) 1.38-1.51 (m, 7H) 1.53-1.68 (m, 11H) 1.71-1.80 (m, 4H)1.97-2.16 (m, 4H) 2.24-2.32 (m, 2H) 2.32-2.42 (m, 1H) 2.43-2.55 (m, 3H)2.56-2.69 (m, 3H) 2.73 (br d, J=2.13 Hz, 1H) 3.07-3.18 (m, 1H) 3.42-3.61(m, 17H) 3.75-3.91 (m, 1H) 4.11-4.24 (m, 1H) 4.43 (s, 2H) 5.59-5.88 (m,4H) 6.06-6.17 (m, 1H) 6.29-6.39 (m, 1H) 7.24-7.39 (m, 2H) 7.52-7.64 (m,2H).

ADL1-D13

Linker-Payload (AD1-D13):4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5ureidopentanamido)benzyl(4-nitrophenyl) carbonate (4.2 mg, 5.693 μmol) in DMF (176 μL, 2.277mmol) was added Hunig's base (2.98 μL, 0.017 mmol). The reaction mixturewas cooled to 0° C., and D13 was added (4.06 mg, 6.262 μmol). Thereaction mixture was stirred at RT until LC/MS showed the reactioncomplete. The reaction mixture was concentrated in vacuo. Flashchromatography of the residue on silica gel with DCM/MeOH gave the titlecompound (4.8 mg, 68% yield). LC/MS (ESI, m/z), 1248.0 [M+H]⁺.

¹H-NMR (400 MHz, CHCl₃-d): δ ppm 0.58-0.98 (m, 10H) 0.99-1.06 (m, 2H)1.11-1.31 (m, 6H) 1.57-1.75 (m, 3H) 2.11-2.26 (m, 1H) 2.30-2.73 (m, 2H)3.28-3.80 (m, 7H) 4.14-4.33 (m, 1H) 4.44-4.55 (m, 1H) 4.63-4.81 (m, 1H)4.92-5.06 (m, 1H) 5.09 (s, 1H) 5.49-5.74 (m, 1H) 5.88-6.09 (m, 1H)6.14-6.36 (m, 1H) 6.99-7.08 (m, 1H) 7.21-7.38 (m, 2H) 7.53-7.67 (m, 1H)7.95 (s, 1H). 1.3.1.6 ADL1-D14

General procedure 1 (outlined in section 1.3.1) was employed tosynthesize1-((2S,3S,6R,7S,10R,E)-2-((E)-1-(1-(1-acetylpiperidin-4-yl)-4-fluoro-1H-benzo[d][1,2,3]triazol-6-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)4-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)piperazine-1,4-dicarboxylate (30 mg, 0.024 mmol, 39.2% yield). LC/MS(ESI, m/z), 1254.5 [M+H]⁺.

¹H-NMR (400 MHz, DMSO-d6): δ ppm 0.75-1.00 (m, 12H), 1.06-1.26 (m, 4H),1.26-1.77 (m, 13H), 1.75-2.02 (m, 7H), 2.08 (s, 8H), 2.23-2.35 (m, 2H),2.54-2.70 (m, 2H), 2.78-3.10 (m, 4H), 3.36 (br s, 13H), 3.66-3.79 (m,1H), 3.95-4.08 (m, 1H), 4.11-4.25 (m, 1H), 4.28-4.46 (m, 1H), 4.46-4.58(m, 1H), 4.58-4.66 (m, 1H), 4.66-4.77 (m, 1H), 5.01 (s, 3H), 5.14-5.27(m, 1H), 5.39 (s, 4H), 5.71-5.79 (m, 1H), 5.87-6.00 (m, 1H), 6.63-6.75(m, 1H), 6.99 (s, 2H), 7.13-7.24 (m, 1H), 7.24-7.34 (m, 2H), 7.52-7.62(m, 2H), 7.65-7.74 (m, 1H), 7.75-7.82 (m, 1H), 7.98-8.13 (m, 1H).

1.3.1.7 ADL1-D33

D33

To a stirred solution of(2S,3S,6R,7S,10R,E)-2-((E)-1-(3-fluoro-5-(4-(2-methoxy-2-oxoethyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (27 mg, 0.042 mmol) in THF/H₂O (3 mL/1 mL) at0° C. was added LiOH, and the reaction mixture was allowed to slowlywarm to 25° C.(2S,3S,6R,7S,10R,E)-2-((E)-1-(3-fluoro-5-(4-(2-methoxy-2-oxoethyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate was synthesized using procedures outlined inInternational Application No. PCT/US2019/026992 (see, e.g., Procedures17 and 19), which is incorporated herein by reference. After 6 hours,the reaction mixture was neutralized with phosphate buffer (NaH₂PO₄, 1.0M, 3 mL) and the phases were separated. The aqueous layer was extractedwith ethyl acetate (3×2 mL), and the combined organic layers were driedwith anhydrous sodium sulfate and concentrated in vacuo. Reversed phasecolumn chromatography afforded the titled compound (19.4 mg, 0.031 mmol,73.4% yield). LC/MS (ESI, m/z), 631.4 [M+H]⁺.

¹H-NMR (400 MHz, CHCl₃-d): δ ppm 0.85-1.16 (m, 2H) 1.23-1.52 (m, 1H)1.57-1.77 (m, 1H) 1.87 (s, 1H) 1.92-2.13 (m, 1H) 2.35-2.55 (m, 1H)2.50-2.74 (m, 1H) 3.11 (br s, 1H) 3.36-3.54 (m, 2H) 3.64 (br d, J=10.42Hz, 2H) 3.77-3.90 (m, 1H) 4.76 (br s, 4H) 5.07-5.19 (m, 1H) 5.38-5.68(m, 1H) 6.40-6.76 (m, 1H) 8.02-8.66 (m, 1H).

ADL1-D33

Linker-Payload (ADL1-D33): To4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (17 mg, 0.023 mmol) in DMF (714 μL, 9.217mmol) was added Hunig's base (12.07 μL, 0.069 mmol). The reactionmixture was cooled to 0° C., and added2-(4-(3-fluoro-5-((E)-2-((2S,3S,6R,7S,10R,E)-10-hydroxy-3,7-dimethyl-12-oxo-6-((piperazine-1-carbonyl)oxy)oxacyclododec-4-en-2-yl)prop-1-en-1-yl)phenyl)piperazin-1-yl)aceticacid (16.71 mg, 0.026 mmol). The reaction mixture was stirred at RTuntil LC/MS showed the reaction complete. The reaction mixture wasconcentrated in vacuo. Flash chromatography on silica gel and furtherHPLC purification gave the titled compound (13.5 mg, 10.98 μmol, 47.7%yield). LC/MS (ESI, m/z), 1229.5 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d₄): δ ppm 0.92-0.99 (m, 10H) 1.01 (d, J=6.78 Hz,3H) 1.24-1.46 (m, 9H) 1.52-1.70 (m, 10H) 1.70-1.82 (m, 1H) 1.87 (d,J=1.00 Hz, 4H) 1.89-2.00 (m, 2H) 2.01-2.23 (m, 2H) 2.27 (t, J=7.40 Hz,2H) 2.46 (dd, J=14.31, 5.27 Hz, 1H) 2.57-2.71 (m, 2H) 2.86 (d, J=0.63Hz, 1H) 3.00 (s, 1H) 3.06-3.27 (m, 4H) 3.35 (s, 5H) 3.41-3.53 (m, 19H)3.66 (s, 2H) 3.76-3.90 (m, 1H) 4.07-4.21 (m, 1H) 4.39-4.69 (m, 37H) 5.09(s, 3H) 5.13 (d, J=10.54 Hz, 1H) 5.52 (dd, J=14.56, 9.03 Hz, 2H) 6.55(br s, 2H) 6.69 (d, J=1.38 Hz, 2H) 7.33 (d, J=8.66 Hz, 2H) 7.59 (d,J=8.53 Hz, 2H) 7.88-8.04 (m, 1H) 8.16-8.33 (m, 1H).

1.3.2 ADL1-D4, ADL1-D5, ADL21-D4, ADL22-D4, ADL23-D4, ADL13-D4 1.3.2.1General Procedure for Preparation of D4 and D5

The 4-step procedure outlined below was employed to synthesize the titlecompounds.

Step 2

To a solution of tri-TES Pladienolide D (1.0 equiv.) in1,2-dichloroethane (0.2 M) at 20° C. was added DMAP (1.5 equiv.),triethylamine (30 equiv.) and 4-Nitrophenyl chloroformate (10 equiv.).The mixture was stirred at 40° C. for 4 days, and then for 2 h at 60° C.The reaction mixture was diluted with EtOAc and washed with water, thenthe layers were separated, and the aqueous layer was extracted withEtOAc (2×). The combined organic extracts were successively washed withwater and brine, dried over magnesium sulfate, filtered and concentratedin vacuo. The resulting residue was purified by silica gelchromatography (EtOAc in Hexane) to afford the intermediate carbonate.Carbonate (1.0 equiv.), DCM (0.2M), triethylamine (3.0 equiv.), andamine (2.0 equiv.) were combined and stirred at RT for 1 hour. Theresulting mixture was concentrated in vacuo and chromatographed(DCM/MeOH) to afford the carbamate intermediate as a mixture ofregioisomers.

Step 3

The mixture of regioisomeric carbamates obtained in Step 2 (1.0 equiv.)was dissolved in DCM (0.04 M). Hunig's base (124 equiv.) was added andthe resulting mixture was cooled to −78° C. HF.Pyridine (30 equiv.) wasadded dropwise and the mixture was warmed to rt and stirred overnight atrt. The mixture was then cooled to −78° C. and saturated sodiumbicarbonate was added dropwise to the reaction mixture and the mixturewas warmed to rt. The organic layers were partioned and the aqueouslayer was extracted with DCM (3×). The combined organic layers weredried over anhydrous sodium sulfate, filtered, and concentrated invacuo. The resulting residue was subjected to reverse-phase HPLCpurification to afford each of desired regioisomeric products.

D5

The title compound was prepared using 2,5-diazabicyclo[2.2.1]heptane instep 2 via the general procedure 1.3.2.1 step 2 & 3

Step 2

A mixture of(2S,3S,6S,7R,10R,E)-7-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-yl2,5-diazabicyclo[2.2.1]heptane-2-carboxylate and(2S,3S,6S,7R,10R,E)-6-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-7-yl2,5-diazabicyclo[2.2.1]heptane-2-carboxylate was obtained (21.7 mg,45.1%) as a Colorless oil. LCMS (ESI, m/z), [M+H]⁺ 1020.6

¹H NMR (400 MHz, METHANOL-d) δ ppm 0.61-0.70 (m, 19H) 0.82-0.95 (m, 10H)0.95-1.04 (m, 29H) 1.23 (br dd, J=7.15, 4.64 Hz, 1H) 1.30 (s, 1H)1.42-1.64 (m, 12H) 1.78 (s, 4H) 1.88-2.01 (m, 2H) 2.06 (br d, J=10.92Hz, 3H) 2.14 (br s, 1H) 2.43 (br d, J=4.64 Hz, 2H) 2.49 (br s, 1H) 2.63(dd, J=8.16, 2.13 Hz, 2H) 2.86-2.91 (m, 1H) 3.47-3.66 (m, 2H) 3.76 (td,J=6.37, 3.33 Hz, 1H) 3.95 (br d, J=4.27 Hz, 1H) 4.34 (br s, 1H) 6.15 (brd, J=10.92 Hz, 1H) 6.52 (dd, J=15.12, 10.98 Hz, 1H)

Step 3 D5

HPLC purification afforded(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl(1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate(5 mg, 34.7%) as a white solid. LCMS (ESI, m/z), [M+H]⁺ 677.6

1H NMR (400 MHz, METHANOL-d4) δ ppm 0.85-0.98 (m, 9H) 1.25 (td, J=7.40,4.14 Hz, 1H) 1.34 (s, 3H) 1.42-1.69 (m, 9H) 1.79 (s, 4H) 1.82-1.96 (m,2H) 2.04 (d, J=9.29 Hz, 3H) 2.33 (br d, J=10.04 Hz, 1H) 2.47-2.54 (m,2H) 2.57-2.72 (m, 2H) 2.87-2.92 (m, 1H) 3.03 (br s, 2H) 3.33-3.43 (m,2H) 3.43-3.57 (m, 2H) 3.77-3.84 (m, 1H) 3.86-3.94 (m, 1H) 4.50 (br s,1H) 4.97-5.11 (m, 2H) 5.60-5.68 (m, 1H) 5.73-5.82 (m, 1H) 5.88 (d,J=15.31 Hz, 1H) 6.15 (br d, J=11.04 Hz, 1H) 6.53 (dd, J=15.25, 10.98 Hz,1H) 8.54 (s, 1H)

D4

The title compound was prepared using piperazine in step 2 via thegeneral procedure 1.3.2.1 step 2 & 3

Step 2:

General procedure 1.3.2.1, step 2 employing piperazine afforded(2S,3S,6S,7R,10R,E)-7-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylpiperazine-1-carboxylate (1.0 g, 0.844 mmol, 81% yield). LC/MS (ESI,m/z), 1008.1 [M+H]⁺.

Step 3: D4

HPLC purification afforded(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (225 mg, 36.8%). LC/MS (ESI, m/z), 665.6[M+H]⁺.

¹H-NMR (400 MHz, CHCl₃-d): δ ppm 0.87-0.92 (m, 6H) 0.94 (t, J=7.40 Hz,3H) 1.16-1.31 (m, 1H) 1.35 (s, 3H) 1.40-1.56 (m, 4H) 1.59 (s, 3H) 1.66(br dd, J=14.68, 7.03 Hz, 3H) 1.76-1.80 (m, 3H) 1.87 (dd, J=14.12, 5.46Hz, 1H) 2.05 (s, 3H) 2.30-2.41 (m, 1H) 2.50 (d, J=3.76 Hz, 2H) 2.56-2.72(m, 2H) 2.90 (br d, J=2.01 Hz, 1H) 3.19 (br t, J=5.14 Hz, 4H) 3.50-3.59(m, 1H) 3.71 (br s, 4H) 3.77-3.89 (m, 1H) 5.01-5.13 (m, 2H) 5.58-5.71(m, 1H) 5.71-5.81 (m, 1H) 5.88 (d, J=15.31 Hz, 1H) 6.15 (br d, J=10.79Hz, 1H) 6.53 (dd, J=15.18, 10.92 Hz, 1H).

ADL1-D4

General procedure 1 (1.3.1) was employed for the preparation of ADL1-D4.Flash chromatography afforded ADL1-D4 (30.5 mg, 0.024 mmol, 77% yield).

LC/MS (ESI, m/z), 1263.8 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d₄): δ ppm 0.87-0.93 (m, 7H) 0.93-1.01 (m, 8H)1.19-1.34 (m, 4H) 1.50 (s, 3H) 1.57 (s, 5H) 1.58-1.70 (m, 6H) 1.70-1.77(m, 1H) 1.78 (s, 3H) 1.83-1.95 (m, 2H) 2.04 (s, 3H) 2.05-2.13 (m, 1H)2.27 (t, J=7.40 Hz, 2H) 2.32-2.42 (m, 1H) 2.50 (d, J=3.64 Hz, 2H)2.55-2.74 (m, 2H) 2.90 (td, J=5.83, 2.26 Hz, 1H) 3.03-3.26 (m, 2H) 3.35(s, 13H) 3.42-3.61 (m, 11H) 3.80 (br dd, J=9.85, 3.58 Hz, 1H) 4.16 (d,J=7.40 Hz, 1H) 4.50 (dd, J=8.91, 5.14 Hz, 1H) 4.56 (s, 1H) 5.05 (dd,J=14.37, 10.10 Hz, 2H) 5.09 (s, 2H) 5.49 (s, 1H) 5.59-5.69 (m, 1H)5.72-5.80 (m, 1H) 5.87 (d, J=15.18 Hz, 1H) 6.09-6.22 (m, 1H) 6.44-6.60(m, 1H) 7.32 (d, J=8.66 Hz, 2H) 7.58 (d, J=8.53 Hz, 2H).

ADL1-D5

General procedure 1 (1.3.1) was employed for the preparation of ADL1-D5using D5. Flash chromatography afforded (14 mg, 51.6% yield). LCMS (ESI,m/z), 1276.43 [M+H]⁺

1H NMR (400 MHz, METHANOL-d4) δ ppm 0.86-1.00 (m, 16H) 1.23-1.40 (m, 7H)1.42-1.69 (m, 16H) 1.73-1.80 (m, 4H) 1.84-1.96 (m, 4H) 1.98-2.10 (m, 4H)2.27 (t, J=7.40 Hz, 2H) 2.31-2.41 (m, 1H) 2.50 (br d, J=3.39 Hz, 2H)2.56-2.64 (m, 1H) 2.64-2.72 (m, 1H) 2.80-2.93 (m, 1H) 3.12 (br d, J=6.90Hz, 1H) 3.16-3.26 (m, 1H) 3.35-3.56 (m, 7H) 3.76-3.86 (m, 1H) 4.16 (d,J=7.40 Hz, 1H) 4.48-4.58 (m, 3H) 4.93-5.16 (m, 4H) 5.65 (br d, J=9.91Hz, 1H) 5.70-5.82 (m, 1H) 5.88 (d, J=15.18 Hz, 1H) 6.14 (br d, J=11.04Hz, 1H) 6.50-6.58 (m, 1H), 6.79 (s, 2H) 7.26-7.36 (m, 2H) 7.56-7.63 (m,2H)

1.3.3 Synthesis of ADL22-D4

To a solution of(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (12 mg, 0.018 mmol) in DMF (2 mL) was added4-((2S,5S)-15-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-10,13-dioxa-3,6-diazapentadecanamido)benzyl(4-nitrophenyl) carbonate (16.98 mg, 0.022 mmol) and Hunig's base (7.00mg, 0.054 mmol). The mixture was stirred for 1 h at 20° C. The mixturewas concentrated in vacuo directly on to silica gel and purified bycolumn chromatography (MeOH/DCM 0-20%) to provide1-((2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)4-(4-((2S,5S)-15-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5-isopropyl-4,7-dioxo-2-(3-ureidopropyl)-10,13-dioxa-3,6-diazapentadecanamido)benzyl)piperazine-1,4-dicarboxylate (12 mg, 9.16 μmol, 50.8% yield) as a whitesolid. LCMS (ESI, m/z), 1310.2 [M+H]⁺

Chemical Formula: C65H96N8O20 Molecular Weight: 1309.52

1H NMR (400 MHz, DMSO-d6) δ ppm 0.73-0.89 (m, 17H) 1.03-1.13 (m, 1H)1.21-1.25 (m, 4H) 1.25-1.40 (m, 5H) 1.42-1.47 (m, 4H) 1.48, (br s, 3H)1.69 (s, 4H) 1.73-1.83 (m, 1H) 1.99 (s, 2H) 2.10-2.25 (m, 1H) 2.36 (brs, 3H) 2.40-2.48 (m, 2H) 2.54-2.66 (m, 2H) 2.72-2.80, (m, 1H) 2.88-3.08(m, 2H) 3.34-3.44 (m, 9H) 3.44-3.48 (m, 3H) 3.48-3.52 (m, 2H) 3.52-3.59(m, 3H) 3.64-3.75 (m, 1H) 4.24 (s, 1H), 4.40 (br d, J=5.65 Hz, 2H) 4.62(d, J=5.02 Hz, 1H) 4.80-4.85 (m, 1H) 4.90 (br d, J=9.03 Hz, 2H) 5.01 (s,2H) 5.40 (s, 2H) 5.48-5.57 (m, 1H), 5.66-5.76 (m, 1H) 5.81-5.91 (m, 1H)5.94-6.01 (m, 1H) 6.02-6.11 (m, 1H) 6.36-6.45 (m, 1H) 7.02 (s, 2H)7.27-7.33 (m, 2H) 7.58 (s, 2H) 7.81-7.89 (m, 1H) 8.11 (br d, J=7.40 Hz,1H) 9.96-10.02 (m, 1H) 1.3.4 General Procedure for synthesis ofADL21-D12, ADL21-D1, ADL21-D4:

Step 1

The Payload (1.0 equiv) and (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-(((S)-4-amino-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1,4-dioxobutan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate(1.0 equiv.) were dissolved in DMF (0.1 M) and Hunig's Base (3.0 euiv.)was added. The reaction mixture was stirred at RT for 30 minutes afterwhich the reaction was concentrated to in vacuo and the resultingresidue was chromatographed (MeOH/DCM) to afford the desired product.

Step 2 and 3

fmoc-Ala-Ala-Aspargine PABC payload (1.0 equiv.), was dissolved inN,N-dimethylformamide (0.05 M), then diethylamine (6.0 equiv.) wasadded. The reaction was stirred at RT for 1 hour and the mixtureevaporated to dryness. The crude product was diluted withN,N-dimethylformamide (0.05 M). 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (1.2 equiv.) andHunig's base (2.5 equiv.) were added to the mixture and stirred at RTfor 1 mixture was then concentrated in vacuo and the resulting residuewas purified via reverse-phour. The phase HPLC to afford the desiredMaleimidocaproyl Ala-Ala-Aspargine PABC linker payload.

1.3.4.1 ADL21-D1

Step 1:

1-(4-((5S,8S,11S)-11-(2-amino-2-oxoethyl)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazadodecan-12-amido)benzyl)4-((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazine-1,4-dicarboxylate (140.2 mg, 71.9%) LCMS (ESI, m/z) 1265.6[M+H]⁺

Chemical Formula: C₆₇H₈₉N₇O₁₇

Molecular Weight: 1264.48

Steps 2 and 3:

1-(4-((S)-4-amino-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)propanamido)-4-oxobutanamido)benzyl)4-((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazine-1,4-dicarboxylate (49.1 mg, 35.8%) LCMS (ESI, m/z) 1258.19[M+Na]⁺

¹H NMR (400 MHz, METHANOL-d4) δ ppm 0.85-0.98 (m, 10H) 1.22 (s, 4H)1.26-1.45 (m, 14H) 1.45-1.69 (m, 12H) 1.79 (s, 3H) 1.90 (s, 2H)2.20-2.27 (m, 2H) 2.52 (br dd, J=10.10, 3.70 Hz, 3H) 2.67 (br d, J=2.13Hz, 1H) 2.85 (t, J=6.15 Hz, 3H) 3.14 (t, J=1.57 Hz, 1H) 3.41-3.57, (m,14H) 3.78-3.88 (m, 1H) 4.27 (dd, J=12.36, 7.09 Hz, 2H) 4.77 (s, 1H)4.97-5.13 (m, 6H) 5.51-5.64 (m, 1H) 5.72 (br d, J=9.66 Hz, 1H), 5.88 (d,J=15.43 Hz, 1H) 6.12-6.17 (m, 1H) 6.47-6.61 (m, 1H) 6.79 (s, 2H) 7.33(m, J=8.66 Hz, 2H), 7.68 (m, J=8.66 Hz, 2H)

1.3.4.2 ADL21-D4 Step 1

1-((2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)4-(4-((5S,8S,11S)-11-(2-amino-2-oxoethyl)-1-(9H-fluoren-9-yl)-5,8-dimethyl-3,6,9-trioxo-2-oxa-4,7,10-triazadodecan-12-amido)benzyl)piperazine-1,4-dicarboxylate (125 mg, 85.6%) LCMS (ESI, m/z) 1293.4[M+H]⁺

Steps 2 and 3

1-((2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)4-(4-((S)-4-amino-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)propanamido)-4-oxobutanamido)benzyl)piperazine-1,4-dicarboxylate (35 mg, 80%) LCMS (ESI, m/z) 1285.0 [M+Na]⁺

¹H NMR (400 MHz, DMSO-d6) δ ppm 0.71-0.87 (m, 9H) 1.04-1.12 (m, 1H)1.14-1.26 (m, 13H) 1.27-1.40 (m, 3H) 1.40-1.52 (m, 9H) 1.52-1.62 (m, 1H)1.69 (s, 3H) 1.74-1.83 (m, 1H) 1.99 (d, J=4.27 Hz, 4H) 2.03-2.14 (m, 2H)2.15-2.26 (m, 1H) 2.31-2.41 (m, 2H) 2.54-2.65 (m, 4H) 2.72-2.80 (m, 1H)3.17 (d, J=5.27 Hz, 3H) 3.36 (br s, 9H) 3.63-3.74 (m, 1H) 3.99-4.12 (m,2H) 4.14-4.22 (m, 1H) 4.23-4.30 (m, 1H) 4.40 (d, J=5.65 Hz, 1H)4.55-4.65 (m, 2H) 4.82 (s, 1H) 4.87-4.93 (m, 2H) 5.01 (s, 2H) 5.47-5.57(m, 1H) 5.66-5.77 (m, 1H) 6.01-6.09 (m, 1H) 6.35-6.45 (m, 1H) 6.89-6.96(m, 1H) 6.99 (s, 2H) 7.30 (d, J=8.66 Hz, 2H) 7.36-7.42 (m, 1H) 7.62 (s,2H) 7.97-8.21 (m, 3H), 9.62-9.72 (m, 1H).

1.3.5 ADL21-D12

To a solution of(2S,3S,6S,7R,10R,E)-7,10-dihydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (50 mg, 0.08 mmol) in DMF (1 mL) was added(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-(((S)-4-amino-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1,4-dioxobutan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate(73.9 mg, 0.096 mmol) followed by N-ethyl-N-isopropylpropan-2-amine (104mg, 0.803 mmol). The reaction mixture was stirred for 1 h. The reactionmixture was concentrated to dryness and purified by silica gel columnchromatography. The fractions containing the desired compound wereconcentrated to provide a clear oil that was carried on directly to thefollowing step assuming 100% yield. The oil obtained was then dissolvedin DMF (2 mL) and charged with diethylamine (58.7 mg, 0.803 mmol). Theresulting mixture was stirred 1 h at 20° C. and concentrated in vacuo.The residue was re-dissolved in DMF (1 mL) and then2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (32.2 mg, 0.104 mmol)and N-ethyl-N-isopropylpropan-2-amine (104 mg, 0.803 mmol) were added.The resulting mixture was stirred for 1 h. The reaction mixture wasconcentrated to dryness and purified under reverse-phase HPLC to furnish1-(4-((S)-4-amino-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)propanamido)-4-oxobutanamido)benzyl)4-((2S,3S,6S,7R,10R,E)-7,10-dihydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazine-1,4-dicarboxylate (7.5 mg, 6.14 μmol, 7.65% yield) as a whitesolid. LCMS (ESI, m/z) 1222.28 [M+H]⁺

1.3.6 ADL21-D8

To a solution of(2S,3S,6S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-7-ylpiperazine-1-carboxylate(27.0 mg, 0.041 mmol) in DMF was added (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-(((S)-4-amino-1-((4((((4nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1,4-dioxobutan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)carbamate(40.5 mg, 0.053 mmol) followed Hunig's base (36.2 μl, 0.203 mmol). Afterstirring for 20 minutes at rt, the mixture was then concentrated invacuo and the resulting residue was purified by column chromatography(0-20% MeOH/DCM) to afford the desired intermediate. The residue wasthen combined with DMF (2 mL) and diethylamine (29.7 mg, 0.406 mmol) andstirred for 20 minutes at rt. Subsequently, the mixture was concentratedin vacuo and the resulting residue was re-dissolved in DMF (2 ml) andtreated with 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (16.28 mg, 0.053 mmol)and Hunig's base (36.2 μl, 0.203 mmol). The resulting mixture wasstirred for 20 minutes before concentrating to dryness and purifying theresulting residue by column chromatography with the intermediate elutingin a 0-20% MeOH/DCM gradient. The residue obtained was then subjected toreverse phase HPLC purification to afford1-((2S,3S,6S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-7-yl)4-(4-((S)-4-amino-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)propanamido)-4-oxobutanamido)benzyl)piperazine-1,4-dicarboxylate (3.5 mg, 2.77 μmol, 6.82% yield) isolatedas a white solid.1-((2S,3S,6S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-7-yl)4-(4-((S)-4-amino-2-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)propanamido)propanamido)-4-oxobutanamido)benzyl)piperazine-1,4-dicarboxylate (3.5 mg, 6.8%) LCMS (ESI, m/z) 1285.60[M+Na]⁺

¹H NMR: 0.68-0.90 (m, 11H) 1.03-1.13 (m, 2H) 1.15-1.26 (m, 14H)1.24-1.38 (m, 4H) 1.40-1.63 (m, 12H), 1.69 (s, 3H) 1.74-1.83 (m, 1H)2.03 (s, 3H) 2.06-2.12 (m, 2H) 2.24-2.41 (m, 3H) 2.53-2.63 (m, 4H),3.79-3.80 (m, 1H) 4.13-4.30 (m, 2H) 4.32-4.33 (m, 1H) 4.33-4.47 (m, 1H)4.44-4.48 (m, 1H) 4.52-4.73 (m, 2H) 4.74-4.97 (m, 2H) 5.03 (s, 3H)5.47-5.61 (m, 1H) 5.65-5.77 (m, 1H) 5.79-5.92 (m, 1H) 5.98-6.12 (m, 1H)6.30-6.46 (m, 1H) 6.75-6.85 (m, 1H) 6.90-6.95 (m, 1H) 6.99 (s, 1H)7.26-7.36 (m, 2H) 7.37-7.46 (m, 1H) 7.56-7.73 (m, 2H) 7.90-8.09 (m, 1H)8.07-8.22 (m, 2H) 8.46 (s, 1H) 9.59-9.78 (m, 1H)

1.3.7 Preparation of DBCO-Val-Cit-pABC Linker-Payload, ADL25-D4

To a solution of3-amino-1-(11,12-dihydrodibenzo[b,f]azocin-5(6H)-yl)propan-1-one (19.36mg, 0.07 mmol) in DCM (5 mL) at 0° C. was added Hunig's base (10.26 mg,0.079 mmol) followed by 4-nitrophenyl carbonochloridate (14.12 mg, 0.07mmol). The reaction was warmed to 20° C. and stirred for 2 h. Themixture was then concentrated to dryness and diluted with DMF. Theresulting mixture was charged Hunig's base (10.26 mg, 0.079 mmol) and1-((2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)4-(4-((S)-2-((S)-2-amino-3-methylbutanamido)-5-ureidopentanamido)benzyl)piperazine-1,4-dicarboxylate (50 mg, 0.047 mmol) and stirred for 1h. Themixture was then concentrated to dryness and purified by columnchromatography followed by reverse-phase purification to afford1-((2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)4-(4-((9S,12S)-9-isopropyl-3,7,10-trioxo-12-(3-ureidopropyl)-2,6,8,11-tetraazatridecan-13-dibenzenecyclooctyneamido)benzyl)piperazine-1,4-dicarboxylate (5.3 mg, 8.27%) LCMS (ESI, m/z) 1372.7 [M]⁺

1.3.8. PREPARATION OF MALEIMIDO-GLU-VAL-CIT-PABC LINKER-PAYLOAD

To a solution of(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (20 mg, 0.03 mmol) in DMF (1 mL) was added(S)-4-(M9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)amino)-5-oxopentanoicacid (32.3 mg, 0.036 mmol) and Hunig's base (11.66 mg, 0.09 mmol) andstirred 1 h at 20° C. Diethylamine (110 mg, 1.504 mmol) was then addedand the mixture was stirred for an additional 30 min at 20° C. Themixture was then diluted with ethyl acetate and concentrated in vacuo.The resulting residue was diluted with DMF (1 mL),2,5-dioxopyrrolidin-1-yl-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate(13.91 mg, 0.045 mmol) and Hunig's base (11.66 mg, 0.09 mmol) and thereaction stirred 50 minutes. The reaction mixture was then diluted withethyl acetate, concentrated to dryness and purified using reverse-phaseHPLC to furnish(S)-5-(((S)-1-(((S)-1-((4-(((4-((((2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazine-1-carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-5-oxopentanoicacid (9.3 mg, 6.68 μmol, 22.20% yield) as a white solid; LCMS (ESI, m/z)1393.4 [M+H]⁺

¹H NMR (400 MHz, DMSO-d6) δ ppm 0.74-0.89 (m, 16H) 1.03-1.66 (m, 25H)1.69 (s, 4H) 1.74-1.94 (m, 2H) 2.00 (s, 4H) 2.09 (br d, J=4.39 Hz, 2H)2.21 (br d, J=7.53 Hz, 3H) 2.31-2.42 (m, 2H) 2.53-2.84 (m, 3H) 2.89-3.09(m, 2H) 3.34-3.48 (m, 10H) 3.65-3.74 (m, 1H) 4.19, (dd, J=8.47, 6.59 Hz,1H) 4.26-4.45 (m, 3H) 4.57-4.64 (m, 1H) 4.79-4.85 (m, 1H) 4.90 (br d,J=9.03 Hz, 2H) 5.01 (s, 2H) 5.43 (br s, 2H), 5.47-5.59 (m, 1H) 5.67-5.80(m, 1H) 5.81-5.93 (m, 1H) 5.94-6.14 (m, 2H) 6.35-6.47 (m, 1H) 6.99 (s,2H) 7.30 (d, J=8.66 Hz, 2H) 7.58, (d, J=8.53 Hz, 2H) 7.63-7.71 (m, 1H)7.98-8.06 (m, 1H) 8.15-8.27 (m, 1H) 9.99-10.09 (m, 1H) 1.3.10. ADL1-D3,ADL10-D3

The payload D3 was prepared via the procedure given below

D3

To a mixture of(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (55 mg, 0.083 mmol) in DCM (3 mL) was added(9H-fluoren-9-yl)methyl (2-oxoethyl)carbamate (46.5 mg, 0.165 mmol) andSodium triacetoxy borohydride (52.6 mg, 0.248 mmol). The reaction wasstirred for 20 minutes at rt. The reaction mixture was then concentratedto dryness and purified by silica gel column chromatography (0-10%MeOH/DCM). The purified material was dissolved in DMF (3 mL). Themixture was then charged with diethylamine (121 mg, 1.655 mmol) andstirred at rt. The mixture was then concentrated in vacuo and purifiedvia reverse phase HPLC purification to afford(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-aminoethyl)piperazine-1-carboxylate (6 mg, 8.48 μmol, 10.25% yield)as a white solid.

LCMS (ESI, m/z), 708.2 [M+H]⁺

¹H NMR (400 MHz, DMSO-d6) δ ppm 0.74-0.86 (m, 9H) 1.04-1.15 (m, 1H) 1.23(s, 3H) 1.26-1.40 (m, 3H) 1.45 (s, 4H) 1.47-1.51 (m, 1H), 1.52-1.63 (m,1H) 1.69 (s, 3H) 1.73-1.82 (m, 1H) 1.99 (s, 3H) 2.13-2.42 (m, 9H)2.53-2.65 (m, 4H) 2.72-2.80 (m, 1H), 3.35-3.41 (m, 3H) 3.65-3.76 (m, 1H)4.36-4.46 (m, 1H) 4.57-4.66 (m, 1H) 4.79-4.85 (m, 1H) 4.87-4.95 (m, 2H)5.43-5.56 (m, 1H) 5.64-5.77 (m, 1H) 5.80-5.91 (m, 1H) 6.01-6.12 (m, 1H)6.33-6.49 (m, 1H).

1.3.10.1 ADL10-D3

To a solution of(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-aminoethyl)piperazine-1-carboxylate (40 mg, 0.057 mmol) in DMF (2mL) was added 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (27.9 mg, 0.09 mmol)followed by Hunig'a base (36.5 mg, 0.283 mmol) and stirred for 20minutes. The resulting mixture was then concentrated onto silica gel andpurified via column chromatography (0-20% MeOH/DCM) to provide(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)ethyl)piperazine-1-carboxylate(22 mg, 43.2% yield) as a yellowish solid. LCMS (ESI, m/z) 902.1 [M+H]⁺

¹H NMR (400 MHz, DMSO-d6) δ ppm 0.73-0.89 (m, 10H) 0.99-1.29 (m, 10H)1.30-1.38 (m, 2H) 1.40-1.49 (m, 9H) 1.52-1.64 (m, 1H), 1.69 (s, 3H)1.74-1.83 (m, 1H) 1.93-2.07 (m, 5H) 2.34 (br d, J=16.94 Hz, 9H) 2.59 (s,3H) 3.07-3.21 (m, 2H) 3.36 (br d, J=14.05 Hz, 3H) 3.66-3.78 (m, 1H)4.37-4.45 (m, 1H) 4.57-4.66 (m, 1H) 4.79-4.86 (m, 1H) 4.87-4.97 (m, 2H)5.46-5.57 (m, 1H) 5.66-5.75 (m, 1H) 5.81-5.91 (m, 1H) 5.99-6.13 (m, 1H)6.31-6.47 (m, 1H) 7.00 (5, 2H) 7.62-7.71 (m, 1H)

1.3.10.2 ADL1-D3

To a solution of(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-aminoethyl)piperazine-1-carboxylate (30 mg, 0.042 mmol) in DMF (2mL) was added (9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-1-oxobutan-2-yl)carbamate(35.7 mg, 0.047 mmol) followed by Hunig's base (16.43 mg, 0.127 mmol)and mixture allowed to stir for 1 h. The resulting mixture wasconcentrated directly on to silica gel and purified by silica gel columnchromatography to provide(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-((((4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)ethyl)piperazine-1-carboxylatewhich was carried on directly to following step directly. To a solutionof(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-((((4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)ethyl)piperazine-1-carboxylate(30 mg, 0.022 mmol) in DMF (2 mL) was added diethylamine (82 mg, 1.123mmol) and mixture allowed to stir 1 h at 20° C. The resulting mixturewas diluted with ethyl acetate and concentrated in vacuo. The resultingresidue was then re-dissolved in DMF and charged with2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (8.31 mg, 0.027 mmol)and Hunig's base (8.71 mg, 0.067 mmol). The resulting mixture stirred 1h at 20° C. The solution was then concentrated on to silica gel andpurified by column chromatography (0-20% MeOH/DCM) to provide(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-((((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)amino)ethyl)piperazine-1-carboxylate(25 mg, 0.019 mmol, 85% yield) as a white solid; LC/MS (ESI, m/z),1306.2 [M]⁺.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 0.75-0.89 (m, 18H) 1.05-1.12 (m, 1H)1.15-1.20 (m, 1H) 1.21-1.26 (m, 4H) 1.26-1.39 (m, 5H) 1.40-1.40 (m, 1H)1.42-1.47 (m, 5H) 1.57 (br s, 3H) 1.69 (s, 4H) 1.73-1.81 (m, 1H)1.92-1.98 (m, 1H) 1.99 (d, J=4.39 Hz, 4H) 2.14-2.26 (m, 1H) 2.29-2.42(m, 4H) 2.42-2.47 (m, 1H) 2.53-2.64 (m, 2H) 2.73-2.78 (m, 1H) 2.88-3.08(m, 2H) 3.35-3.60 (m, 19H) 3.65-3.75 (m, 1H) 4.22 (dd, J=8.47, 6.71 Hz,1H) 4.32-4.45 (m, 2H) 4.60-4.65 (m, 1H) 4.79-4.85 (m, 1H) 4.90 (br d,J=9.03 Hz, 2H) 5.01 (s, 2H) 5.40 (s, 2H) 5.47-5.58 (m, 1H) 5.66-5.77 (m,1H) 5.87 (s, 1H) 5.97 (s, 1H) 6.03-6.13 (m, 1H) 6.35-6.45 (m, 1H) 7.02(s, 2H) 7.30 (d, J=8.53 Hz, 2H) 7.59 (d, J=8.53 Hz, 2H) 7.81-7.88 (m,1H), 8.09-8.15 (m, 1H) 9.90-10.09 (m, 1H)

1.3.11 ADL1-D6 and ADL1-D7 were Prepared Via the General Procedure Below

To a solution of(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (30 mg, 0.045 mmol) in DCM (0.1 M) was added(9H-fluoren-9-yl)methyl methyl(2-oxoethyl)carbamate (5 equiv.) andsodium triacetoxy borohydride (10 equiv.) and stirred for 30 minutes.The mixture then diluted with ethyl acetate and washed with water andbrine. The organic layer was dried over anhydrous Na₂SO₄, concentratedand purified by column chromatography (0-10% MeOH/DCM gradient) toisolate the desired product. The resulting material was then dilutedwith DMF (0.1M), charged with diethylamine (20 equiv.) and stirred for20 minutes. The mixture was then concentrated to dryness and dilutedagain in DMF and charged with4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (1.2 equiv.), then Hunig's base (3.0 equiv.).The mixture was stirred for 30 minutes, concentrated in vacuo, andpurified via preparative HPLC purification to afford target product.

1.3.11.2 ADL1-D7

General procedure 1 (1.3.11) afforded(2S,3S,6S,7R,10R,E)-7,10-dihydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-(((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)phenoxy)carbonyl)(methyl)amino)ethyl)piperazine-1-carboxylate(3.1 mg, 5.03% yield). LC/MS (ESI, m/z), 1278.4 [M+H]⁺.

1.3.11.2 ADL1-D6

General procedure 1 (1.3.11) afforded(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-(((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)phenoxy)carbonyl)(methyl)amino)ethyl)piperazine-1-carboxylate(2 mg, 3.36% yield LC/MS (ESI, m/z), 1321.37 [M+H]⁺.

1.3.1.5 ADL1-D9, ADL6-D9, & ADL1-D13 D9 & D13

D9 and D13 were synthesized as a 3:1 mixture of isomers employingprocedures outlined in the synthesis of D4 (Scheme 4) utilizing tri-TESPladienolide B.

Payload (D9): LC/MS (ESI, m/z), 649.7 [M+H]⁺.

¹H-NMR (400 MHz, CHCl₃-d): δ ppm 0.87-1.01 (m, 10H) 1.08 (d, J=6.78 Hz,3H) 1.27-1.63 (m, 12H) 1.66-1.74 (m, 1H) 1.76 (s, 3H) 1.97-2.10 (m, 3H)2.35-2.57 (m, 5H) 2.58-2.65 (m, 1H) 2.65-2.71 (m, 1H) 2.77 (td, J=5.93,2.32 Hz, 1H) 2.89-3.05 (m, 4H) 3.07-3.34 (m, 8H) 3.50-3.68 (m, 5H)3.72-3.88 (m, 1H) 4.88-5.09 (m, 1H) 5.18 (d, J=10.67 Hz, 1H) 5.50-5.84(m, 3H) 6.01-6.13 (m, 1H) 6.19-6.36 (m, 1H).

Payload (D13): LC/MS (ESI, m/z), 649.6 [M+H]⁺.

¹H NMR (400 MHz, CHCl₃-d) δ ppm 0.84-1.01 (m, 11H) 1.08 (d, J=6.78 Hz,3H) 1.29-1.64 (m, 10H) 1.63-1.73 (m, 1H) 1.76 (s, 3H) 1.94-2.12 (m, 4H)2.37-2.58 (m, 4H) 2.59-2.65 (m, 1H) 2.68 (dd, J=7.40, 2.26 Hz, 1H) 2.77(td, J=5.93, 2.32 Hz, 1H) 3.01-3.30 (m, 4H) 3.49 (s, 1H) 3.54-3.67 (m,2H) 3.69-3.92 (m, 5H) 4.13-4.78 (m, 11H) 5.13-5.24 (m, 2H) 5.48-5.61 (m,1H) 5.62-5.74 (m, 2H) 6.04-6.13 (m, 1H) 6.18-6.32 (m, 1H).

ADL1-D9

General procedure 1 (outlined in section 1.3.1) was employed tosynthesize ADL1-D9.

Linker-Payload (ADL1-D9): (30.5 mg, 0.024 mmol, 77% yield). LC/MS (ESI,m/z), 1263.8 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d₄): δ ppm 0.87-0.93 (m, 7H) 0.93-1.01 (m, 8H)1.19-1.34 (m, 4H) 1.50 (s, 3H) 1.57 (s, 5H) 1.58-1.70 (m, 6H) 1.70-1.77(m, 1H) 1.78 (s, 3H) 1.83-1.95 (m, 2H) 2.04 (s, 3H) 2.05-2.13 (m, 1H)2.27 (t, J=7.40 Hz, 2H) 2.32-2.42 (m, 1H) 2.50 (d, J=3.64 Hz, 2H)2.55-2.74 (m, 2H) 2.90 (td, J=5.83, 2.26 Hz, 1H) 3.03-3.26 (m, 2H) 3.35(s, 13H) 3.42-3.61 (m, 11H) 3.80 (br dd, J=9.85, 3.58 Hz, 1H) 4.16 (d,J=7.40 Hz, 1H) 4.50 (dd, J=8.91, 5.14 Hz, 1H) 4.56 (s, 1H) 5.05 (dd,J=14.37, 10.10 Hz, 2H) 5.09 (s, 2H) 5.49 (s, 1H) 5.59-5.69 (m, 1H)5.72-5.80 (m, 1H) 5.87 (d, J=15.18 Hz, 1H) 6.09-6.22 (m, 1H) 6.44-6.60(m, 1H) 7.32 (d, J=8.66 Hz, 2H) 7.58 (d, J=8.53 Hz, 2H).

ADL6-D9

General procedure 1 (outlined in section 1.3.1) was employed tosynthesize ADL6-D9.

Linker-Payload (ADL6-D9): To diluted4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl(4-nitrophenyl) carbonate (16.5 mg, 0.025 mmol) in DMF (784 μL) wasadded Hunig's Base (13.27 μL, 0.076 mmol). The reaction mixture wascooled to 0° C., and D9 was added. The reaction mixture was stirred atRT until LC/MS showed the reaction complete. The reaction mixture wasconcentrated in vacuo. Flash chromatography of the residue on silica gelwith DCM/MeOH gave the titled compound (16.7 mg, 57% yield). LC/MS (ESI,m/z), 1184.6 [M+Na]⁺.

¹H-NMR (400 MHz, MeOH-d₄): δ ppm 0.80-1.02 (m, 16H) 1.05-1.12 (m, 4H)1.13-1.34 (m, 5H) 1.38-1.51 (m, 7H) 1.53-1.68 (m, 11H) 1.71-1.80 (m, 4H)1.97-2.16 (m, 4H) 2.24-2.32 (m, 2H) 2.32-2.42 (m, 1H) 2.43-2.55 (m, 3H)2.56-2.69 (m, 3H) 2.73 (br d, J=2.13 Hz, 1H) 3.07-3.18 (m, 1H) 3.42-3.61(m, 17H) 3.75-3.91 (m, 1H) 4.11-4.24 (m, 1H) 4.43 (s, 2H) 5.59-5.88 (m,4H) 6.06-6.17 (m, 1H) 6.29-6.39 (m, 1H) 7.24-7.39 (m, 2H) 7.52-7.64 (m,2H).

ADL1-D13

Linker-Payload (AD1-D13):4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5ureidopentanamido)benzyl(4-nitrophenyl) carbonate (4.2 mg, 5.693 μmol) in DMF (176 μL, 2.277mmol) was added Hunig's base (2.98 μL, 0.017 mmol). The reaction mixturewas cooled to 0° C., and D13 was added (4.06 mg, 6.262 μmol). Thereaction mixture was stirred at RT until LC/MS showed the reaction wascomplete. The reaction mixture was concentrated in vacuo. Flashchromatography of the residue on silica gel with DCM/MeOH gave thetitled compound (4.8 mg, 68% yield). LC/MS (ESI, m/z), 1248.0 [M+H]⁺.

¹H-NMR (400 MHz, CHCl₃-d): δ ppm 0.58-0.98 (m, 10H) 0.99-1.06 (m, 2H)1.11-1.31 (m, 6H) 1.57-1.75 (m, 3H) 2.11-2.26 (m, 1H) 2.30-2.73 (m, 2H)3.28-3.80 (m, 7H) 4.14-4.33 (m, 1H) 4.44-4.55 (m, 1H) 4.63-4.81 (m, 1H)4.92-5.06 (m, 1H) 5.09 (s, 1H) 5.49-5.74 (m, 1H) 5.88-6.09 (m, 1H)6.14-6.36 (m, 1H) 6.99-7.08 (m, 1H) 7.21-7.38 (m, 2H) 7.53-7.67 (m, 1H)7.95 (s, 1H).

1.3.1.6 ADL1-D14

General procedure 1 (outlined in section 1.3.1) was employed tosynthesize1-((2S,3S,6R,7S,10R,E)-2-((E)-1-(1-(1-acetylpiperidin-4-yl)-4-fluoro-1H-benzo[d][1,2,3]triazol-6-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)4-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)piperazine-1,4-dicarboxylate (30 mg, 0.024 mmol, 39.2% yield). LC/MS(ESI, m/z), 1254.5 [M+H]⁺.

¹H-NMR (400 MHz, DMSO-d₆): δ ppm 0.75-1.00 (m, 12H), 1.06-1.26 (m, 4H),1.26-1.77 (m, 13H), 1.75-2.02 (m, 7H), 2.08 (s, 8H), 2.23-2.35 (m, 2H),2.54-2.70 (m, 2H), 2.78-3.10 (m, 4H), 3.36 (br s, 13H), 3.66-3.79 (m,1H), 3.95-4.08 (m, 1H), 4.11-4.25 (m, 1H), 4.28-4.46 (m, 1H), 4.46-4.58(m, 1H), 4.58-4.66 (m, 1H), 4.66-4.77 (m, 1H), 5.01 (s, 3H), 5.14-5.27(m, 1H), 5.39 (s, 4H), 5.71-5.79 (m, 1H), 5.87-6.00 (m, 1H), 6.63-6.75(m, 1H), 6.99 (s, 2H), 7.13-7.24 (m, 1H), 7.24-7.34 (m, 2H), 7.52-7.62(m, 2H), 7.65-7.74 (m, 1H), 7.75-7.82 (m, 1H), 7.98-8.13 (m, 1H).

1.3.1.7 ADL1-D33 D33

To a stirred solution of(2S,3S,6R,7S,10R,E)-2-((E)-1-(3-fluoro-5-(4-(2-methoxy-2-oxoethyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (27 mg, 0.042 mmol) in THF/H₂O (3 mL/1 mL) at0° C. was added LiOH, and the reaction mixture was allowed to slowlywarm to 25° C.(2S,3S,6R,7S,10R,E)-2-((E)-1-(3-fluoro-5-(4-(2-methoxy-2-oxoethyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate was synthesized using procedures outlined inInternational Application No. PCT/US2019/026992 (see, e.g., Procedures17 and 19), which is incorporated herein by reference. After 6 hours,the reaction mixture was neutralized with phosphate buffer (NaH₂PO₄, 1.0M, 3 mL) and the phases were separated. The aqueous layer was extractedwith ethyl acetate (3×2 mL), and the combined organic layers were driedwith anhydrous sodium sulfate and concentrated in vacuo. Reversed phasecolumn chromatography afforded the titled compound (19.4 mg, 0.031 mmol,73.4% yield). LC/MS (ESI, m/z), 631.4 [M+H]⁺.

¹H-NMR (400 MHz, CHCl₃-d): δ ppm 0.85-1.16 (m, 2H) 1.23-1.52 (m, 1H)1.57-1.77 (m, 1H) 1.87 (s, 1H) 1.92-2.13 (m, 1H) 2.35-2.55 (m, 1H)2.50-2.74 (m, 1H) 3.11 (br s, 1H) 3.36-3.54 (m, 2H) 3.64 (br d, J=10.42Hz, 2H) 3.77-3.90 (m, 1H) 4.76 (br s, 4H) 5.07-5.19 (m, 1H) 5.38-5.68(m, 1H) 6.40-6.76 (m, 1H) 8.02-8.66 (m, 1H).

ADL1-D33

Linker-Payload (ADL1-D33): To4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (17 mg, 0.023 mmol) in DMF (714 μL, 9.217mmol) was added Hunig's base (12.07 μL, 0.069 mmol). The reactionmixture was cooled to 0° C., and added2-(4-(3-fluoro-5-((E)-2-((2S,3S,6R,7S,10R,E)-10-hydroxy-3,7-dimethyl-12-oxo-6-((piperazine-1-carbonyl)oxy)oxacyclododec-4-en-2-yl)prop-1-en-1-yl)phenyl)piperazin-1-yl)aceticacid (16.71 mg, 0.026 mmol). The reaction mixture was stirred at RTuntil LC/MS showed the reaction was complete. The reaction mixture wasthen concentrated in vacuo. Flash chromatography on silica gel andfurther HPLC purification gave the titled compound (13.5 mg, 10.98 μmol,47.7% yield). LC/MS (ESI, m/z), 1229.5 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d₄): δ ppm 0.92-0.99 (m, 10H) 1.01 (d, J=6.78 Hz,3H) 1.24-1.46 (m, 9H) 1.52-1.70 (m, 10H) 1.70-1.82 (m, 1H) 1.87 (d,J=1.00 Hz, 4H) 1.89-2.00 (m, 2H) 2.01-2.23 (m, 2H) 2.27 (t, J=7.40 Hz,2H) 2.46 (dd, J=14.31, 5.27 Hz, 1H) 2.57-2.71 (m, 2H) 2.86 (d, J=0.63Hz, 1H) 3.00 (s, 1H) 3.06-3.27 (m, 4H) 3.35 (s, 5H) 3.41-3.53 (m, 19H)3.66 (s, 2H) 3.76-3.90 (m, 1H) 4.07-4.21 (m, 1H) 4.39-4.69 (m, 37H) 5.09(s, 3H) 5.13 (d, J=10.54 Hz, 1H) 5.52 (dd, J=14.56, 9.03 Hz, 2H) 6.55(br s, 2H) 6.69 (d, J=1.38 Hz, 2H) 7.33 (d, J=8.66 Hz, 2H) 7.59 (d,J=8.53 Hz, 2H) 7.88-8.04 (m, 1H) 8.16-8.33 (m, 1H).

ADL1-D28

4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (6.17 mg, 8.37 μmol) and(2S,3S,6R,7S,10R,E)-2-((E)-1-(3-fluoro-5-(4-((1-methyl-1H-imidazol-4-yl)sulfonyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (6 mg, 8.37 μmol) were dissolved in DMF (0.3ml) and Hunig's Base (4.39 μL, 0.025 mmol) was added. The reactionmixture was stirred at rt overnight. The solvent was evaporated andsubjected to reverse-phase HPLC purification to afford1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)4-((2S,3S,6R,7S,10R,E)-2-((E)-1-(3-fluoro-5-(4-((1-methyl-1H-pyrazol-4-yl)sulfonyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazine-1,4-dicarboxylate (1.5 mg, 13.6%). LC/MS (ESI, m/z), 1315.66[M+H]⁺.

¹H NMR (400 MHz, METHANOL-d4) δ ppm 0.85-0.93 (m, 12H) 1.16-1.31 (m, 6H)1.43-1.57 (m, 9H) 1.62-1.70 (m, 1H) 1.75 (s, 3H) 1.77-1.90 (m, 2H)1.96-2.03 (m, 1H) 2.13-2.20 (m, 2H) 2.31-2.40 (m, 1H) 2.46-2.55 (m, 2H)2.97-3.06 (m, 2H) 3.08-3.10 (m, 1H) 3.15 (br d, J=6.27 Hz, 9H) 3.33-3.43(m, 10H) 3.71 (s, 4H) 4.03-4.09 (m, 1H) 4.37-4.44 (m, 1H) 4.44-4.53 (m,1H) 4.96-5.06 (m, 3H) 5.33-5.51 (m, 2H) 6.36-6.44 (m, 2H) 6.45-6.53 (m,2H) 6.69 (s, 2H) 7.19-7.25 (m, 2H) 7.47-7.52 (m, 2H) 7.63-7.70 (m, 2H)

ADL1-D31

4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (13.12 mg, 0.018 mmol) and(2S,3S,6R,7S,10R,E)-2-((E)-1-(5-chloropyridin-3-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (9 mg, 0.018 mmol) were dissolved in DMF (179μl, 2.312 mmol) and Hunig's Base (9.32 μl, 0.053 mmol) was added. Thereaction mixture was stirred at rt overnight. The solvent was evaporatedand purified via reverse-phase HPLC to afford1-((2S,3S,6R,7S,10R,E)-2-((E)-1-(5-chloropyridin-3-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)4-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)piperazine-1,4-dicarboxylate (6.9 mg, 34.09%). LC/MS (ESI, m/z), 1105.59[M+H]⁺.

1H NMR (400 MHz, METHANOL-d4) δ ppm 0.88 (s, 12H) 1.17-1.31 (m, 4H)1.43-1.57 (m, 8H) 1.61-1.70 (m, 1H) 1.80 (d, J=1.25 Hz, 5H), 1.92-2.01(m, 1H) 2.14-2.20 (m, 2H) 2.32-2.42 (m, 1H) 2.48-2.59 (m, 2H) 2.96-3.14(m, 3H) 3.38 (s, 10H) 3.36-3.36 (m, 1H) 3.38-3.40 (m, 3H) 3.67-3.75 (m,1H) 4.03-4.07 (m, 1H) 4.37-4.42 (m, 1H) 4.97-5.01 (m, 2H) 5.04-5.11 (m,1H) 5.35-5.51 (m, 2H) 6.46-6.52, (m, 1H) 6.69 (s, 2H) 7.18-7.26 (m, 2H)7.45-7.54 (m, 2H) 7.68-7.74 (m, 1H) 8.29-8.34 (m, 2H).

ADL1-D29

4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (8.62 mg, 0.012 mmol) and(2S,3S,6R,7S,10R,E)-2-((E)-1-(3-(dimethylamino)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (6 mg, 0.012 mmol) were dissolved in DMF (118μl, 1.518 mmol) and Hunig's Base (6.12 μl, 0.035 mmol). The reactionmixture was stirred at rt overnight. The solvent was evaporated andpurified via reverse-phase HPLC to afford1-((2S,3S,6R,7S,10R,E)-2-((E)-1-(3-(dimethylamino)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)4-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)piperazine-1,4-dicarboxylate (4.0 mg, 30.79%). LC/MS (ESI, m/z), 1112.59[M+H]⁺.

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 0.87 (s, 12H) 1.17-1.23 (m, 3H)1.26-1.32 (m, 1H) 1.44-1.57 (m, 8H) 1.63-1.69 (m, 1H) 1.74-1.81 (m, 4H)1.83-1.88 (m, 1H) 1.93-2.03 (m, 1H) 2.13-2.20 (m, 2H) 2.34-2.42 (m, 1H)2.48-2.57 (m, 2H) 2.81 (s, 5H) 2.98-3.05 (m, 2H) 3.08-3.14 (m, 1H) 3.38(br s, 9H) 3.65-3.75 (m, 1H) 4.03-4.07 (m, 1H) 4.35-4.43 (m, 1H)4.44-4.52 (m, 1H) 4.96-5.01 (m, 2H) 5.02-5.08 (m, 1H) 5.34-5.51 (m, 2H)6.43-6.48 (m, 1H) 6.51-6.61 (m, 3H) 6.69 (s, 2H) 7.03-7.10 (m, 1H)7.20-7.26 (m, 2H) 7.46-7.51 (m, 2H).

ADL1-D35

4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (9.78 mg, 0.013 mmol) and(2S,3S,6R,7S,10R,E)-10-hydroxy-3,7-dimethyl-12-oxo-2-((E)-1-(3-(pyrrolidin-1-ylsulfonyl)phenyl)prop-1-en-2-yl)oxacyclododec-4-en-6-ylpiperazine-1-carboxylate (8 mg, 0.013 mmol) were dissolved in DMF (133μl, 1.722 mmol) and Hunig's Base (6.94 μl, 0.04 mmol) [two drops] wasadded. The resulting mixture was stirred at rt overnight, concentratedin vacuo, and purified via reverse-phase HPLC to afford product1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)4-((2S,3S,6R,7S,10R,E)-10-hydroxy-3,7-dimethyl-12-oxo-2-((E)-1-(3-(pyrrolidin-1-ylsulfonyl)phenyl)prop-1-en-2-yl)oxacyclododec-4-en-6-yl)piperazine-1,4-dicarboxylate (8.2 mg, 51.47%). LC/MS (ESI, m/z), 1203.53[M+H]⁺.

1H NMR (400 MHz, METHANOL-d4) δ ppm 0.85-0.93 (m, 12H) 1.17-1.34 (m, 4H)1.42-1.57 (m, 8H) 1.61-1.69 (m, 5H) 1.79 (s, 4H) 1.83-1.90 (m, 1H)1.94-2.03 (m, 1H) 2.12-2.20 (m, 2H) 2.33-2.43 (m, 1H) 2.48-2.59 (m, 2H)2.96-3.05 (m, 2H) 3.06-3.17 (m, 6H) 3.31-3.41 (m, 10H) 3.67-3.77 (m, 1H)4.02-4.10 (m, 1H) 4.36-4.43 (m, 1H) 4.96-5.02 (m, 2H) 5.04-5.11 (m, 1H)5.35-5.51 (m, 2H) 6.59 (s, 1H) 6.69 (s, 2H) 7.20-7.27 (m, 2H) 7.45-7.52(m, 4H) 7.58-7.65 (m, 2H).

1.4 Preparation of MC-Val-Ala-pABC Linker-Payloads 1.4.1 Overview

Step 1: Macrocycle payload (1.0 equiv.), DMF (0.1 M),(9H-fluoren-9-yl)methyl((S)-3-methyl-1-(((S)-1-((4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-1-oxobutan-2-yl)carbamate(1.5 equiv.; prepared using the procedure described in WO 2012/153193),and Hunig's base (3.0 equiv.) were combined and stirred overnight. Thereaction mix was concentrated to dryness and chromatographed to affordfmoc Val-Ala-pABC-payload.

Step 2: Ammonium fmoc-Val-Ala-pABC payload (1.0 equiv.), dissolved inDMF (0.1 M), and diethylamine (10 equiv.) were combined and stirred for30 min. The reaction mix was concentrated to dryness under high vacuum.The crude product was dissolved in DMF (0.1 M), and Hunig's base (2equiv.) was added, after which 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (3 equiv.) was addedand stirred overnight. The reaction mix was then concentrated to drynessand chromatographed (0-30% MeOH in DCM) to afford the desiredMC-Val-Ala-pABC-payload.

1.4.1.1 ADL6-D1

Scheme 5—Step 1: General procedure outlined above (section 1.4.1) wasfollowed to afford1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)4-((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazine-1,4-dicarboxylate (32 mg, 0.027 mmol, 27.9% yield). LC/MS(ESI, m/z), [M+Na]⁺ 1200.5.

Scheme 5—Step 2: General procedure outlined above (section 1.4.1) wasfollowed to afford1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)4-((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)piperazine-1,4-dicarboxylate (24 mg, 0.021 mmol, 77% yield). LC/MS (ESI,m/z), 1149.2 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.85-1.02 (m, 16H), 1.20-1.23 (m, 3H)1.23-1.32 (m, 3H) 1.34 (s, 3H) 1.41-1.46 (m, 4H) 1.46-1.72 (m, 11H) 1.78(s, 3H) 1.87 (dd, J=14.05, 5.40 Hz, 1H) 2.08 (dq, J=13.77, 6.83 Hz, 1H)2.27 (t, J=7.40 Hz, 2H) 2.43-2.63 (m, 3H) 2.64-2.70 (m, 1H) 2.90 (td,J=5.83, 2.26 Hz, 1H) 3.32-3.33 (m, 4H) 3.42-3.57 (m, 11H) 3.82 (br dd,J=8.66, 3.89 Hz, 1H) 4.15 (d, J=7.15 Hz, 1H), 4.47 (q, J=7.15 Hz, 1H)4.99-5.12 (m, 4H) 5.57 (dd, J=15.18, 9.79 Hz, 1H) 5.69-5.78 (m, 1H) 5.87(d, J=15.18 Hz, 1H) 6.13 (d, J=10.92 Hz, 1H), 6.53 (dd, J=15.31, 10.92Hz, 1H) 6.78 (s, 2H) 7.32 (d, J=8.53 Hz, 2H) 7.58 (d, J=8.53 Hz, 2H).

1.4.1.2 ADL6-D16

Scheme 5—Step 1: General procedure outlined above (section 1.4.1) wasfollowed to afford1-((2S,3S,6R,7S,10R,E)-2-((E)-1-(3-(4-(2-(1H-pyrazol-1-yl)acetyl)piperazin-1-yl)-5-fluorophenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)4-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)piperazine-1,4-dicarboxylate (0.060 g, 0.049 mmol, 60.8% yield). LC/MS(ESI, m/z), 1223.68 [M+H].

Scheme 5—Step 2: General procedure outlined above (section 1.4.1) wasfollowed to afford1-((2S,3S,6R,7S,10R,E)-2-((E)-1-(3-(4-(2-(1H-pyrazol-1-yl)acetyl)piperazin-1-yl)-5-fluorophenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)4-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)piperazine-1,4-dicarboxylate (11.50 mg, 9.64 μmol, 12.05% yield). LC/MS(ESI, m/z),1194.82 [M+H]⁺.

¹H-NMR (400 MHz, CHCl₃-d): δ ppm 0.97 (br d, J=1.25 Hz, 12H), 1.24-1.41(m, 5H), 1.41-1.47 (m, 3H), 1.52-1.69 (m, 6H), 1.84-1.90 (m, 3H),1.92-2.01 (m, 1H), 2.02-2.16 (m, 1H), 2.23-2.30 (m, 2H), 2.42-2.51 (m,1H), 2.57-2.65 (m, 2H), 3.19-3.28 (m, 4H), 3.35 (s, 4H), 3.40-3.54 (m,11H), 3.69-3.77 (m, 4H), 3.78-3.85 (m, 1H), 4.12-4.18 (m, 1H), 4.43-4.51(m, 1H), 5.07-5.16 (m, 3H), 5.20 (s, 2H), 5.42-5.62 (m, 2H), 6.29-6.38(m, 1H), 6.46-6.58 (m, 2H), 6.59-6.68 (m, 2H), 7.28-7.37 (m, 2H),7.50-7.67 (m, 4H).

1.5 Preparation of MC-Val-Ala-pAB Linker-Payloads 1.5.1 Overview

Step 1: (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(0.5 g, 0.97 mmol), 1,4-dioxane (9.70 mL, 0.97 mmol), triphenylphosphine(0.509 g, 1.939 mmol), N-bromosuccinimide (0.345 g, 1.939 mmol), and DMF(2.424 mL, 0.97 mmol) were combined and stirred for 3 hours at RT. Thereaction mix was concentrated and chromatographed to afford(9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(bromomethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(116 mg, 0.201 mmol, 20.68% yield). LC/MS (ESI, m/z), 580.14 [M+H].

Step 2: The payload (1.0 equiv.), (9H-fluoren-9-yl)methyl((S)-1-(((S)-1-((4-(bromomethyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(0.069 g, 0.12 mmol), N,N-dimethylformamide (0.1 M), and Hunig's base(1.5 equiv.) were combined and stirred overnight. The reaction mix wasconcentrated to dryness and chromatographed (0-10% MeOH in DCM) toafford the quaternary ammonium fmoc-Val-Ala-pAB payload.

Step 3: Quaternary ammonium fmoc-Val-Ala-pAB payload (1.0 equiv.),dissolved in DMF (0.1 M), and diethylamine (10 equiv.) were combined andstirred for 30 min. The reaction mix was concentrated to dryness underhigh vacuum. The crude product was dissolved in DMF (0.1 M) and Hunig'sbase (2 equiv.) was added, after which 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (3 equiv.) was addedand stirred overnight. The reaction mix was then concentrated to drynessand chromatographed (0-30% MeOH in DCM) to afford the desired quaternaryammonium linker-payload.

1.5.1.1 ADL5-D2

Scheme 6—Step 2:1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-iumbromide (30 mg, 0.024 mmol, 26.5% yield). LC/MS (ESI, m/z), 1149.77[M+H]⁺.

Scheme 6—Step 3:mono(1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium-2-ylium)monobromide (4.8 mg, 15.33% yield). LC/MS (ESI, m/z), 1120.99 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.88 (s, 15H), 1.09-1.26 (m, 9H),1.31-1.58 (m, 14H), 1.42-1.44 (m, 1H), 1.68 (s, 3H), 1.73-1.81 (m, 1H),1.91-2.03 (m, 1H), 2.14-2.22 (m, 2H), 2.35-2.52 (m, 3H), 2.53-2.60 (m,1H), 2.74-2.83 (m, 1H), 2.97 (s, 3H), 3.25 (s, 1H), 3.28-3.46 (m, 8H),3.52-3.66 (m, 2H), 3.70-3.78 (m, 1H), 3.97-4.11 (m, 3H), 4.32-4.41 (m,1H), 4.45-4.54 (m, 2H), 4.91-5.01 (m, 2H), 5.44-5.55 (m, 1H), 5.57-5.70(m, 1H), 5.72-5.82 (m, 1H), 6.00-6.08 (m, 1H), 6.43 (dd, J=15.25, 10.98Hz, 1H), 6.69 (s, 2H), 7.36-7.47 (m, 2H), 7.67-7.75 (m, 2H).

1.5.1.2 ADL5-D19

General procedure outlined above (section 1.5.1) was employed tosynthesize ADL5-D19. The payload D19 was synthesized using proceduresoutlined in section 1.2.1.

D19

(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((S,2E,4E)-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-methylpiperazine-1-carboxylate (114.2 mg, 0.180 mmol, 82% yield).LC/MS (ESI, m/z), 635.8 [M+H].

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.88-1.00 (m, 9H) 1.10 (d, J=6.65 Hz,3H) 1.16-1.27 (m, 4H) 1.40-1.70 (m, 8H) 1.77 (d, J=0.88 Hz, 3H)2.28-2.36 (m, 3H) 2.42 (br t, J=5.08 Hz, 3H) 2.47-2.61 (m, 4H) 2.68 (dd,J=8.22, 2.20 Hz, 1H) 2.74 (td, J=5.99, 2.20 Hz, 1H) 3.13-3.17 (m, 1H)3.34-3.38 (m, 3H) 3.47-3.58 (m, 5H) 3.81-3.87 (m, 1H) 5.01-5.10 (m, 2H)5.54-5.81 (m, 3H) 6.06-6.15 (m, 1H) 6.34 (dd, J=15.00, 10.98 Hz, 1H).

1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((S,2E,4E)-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium(25 mg, 45.6%). LC/MS (ESI, m/z), 1133.1 [M+]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.94 (br dd, J=18.45, 7.15 Hz, 18H)1.10 (d, J=6.78 Hz, 3H) 1.25 (s, 4H) 1.28-1.35 (m, 1H) 1.46 (br d,J=7.03 Hz, 9H) 1.59-1.70 (m, 2H) 1.77 (s, 3H) 2.05-2.12 (m, 1H)2.44-2.60 (m, 4H) 2.63-2.72 (m, 1H) 2.70-2.77 (m, 1H) 3.06 (s, 3H)3.40-3.44 (m, 1H) 3.50 (br d, J=1.63 Hz, 4H) 3.64-3.75 (m, 1H) 3.80-3.88(m, 1H) 3.91-3.99 (m, 1H) 4.04-4.20 (m, 1H) 4.21-4.28 (m, 1H) 4.37-4.43(m, 1H) 4.59 (br s, 2H) 4.93-4.95 (m, 2H) 5.06-5.08 (m, 2H) 5.57-5.80(m, 4H) 6.05-6.16 (m, 1H) 6.27-6.41 (m, 1H) 7.28-7.37 (m, 2H) 7.38-7.45(m, 2H) 7.47-7.53 (m, 2H) 7.65-7.72 (m, 2H) 7.74-7.86 (m, 4H).

ADL5-D19

Linker-Payload (ADL5-D19):1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((S,2E,4E)-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium(19.4 mg, 0.018 mmol, 80% yield). LC/MS (ESI, m/z), 1105.9 [M+H].

¹H NMR (400 MHz, MeOH-d4): δ ppm 0.85-1.03 (m, 16H) 1.10 (d, J=6.78 Hz,3H) 1.17-1.27 (m, 4H) 1.29-1.37 (m, 3H) 1.44-1.70, (m, 15H) 1.74-1.80(m, 3H) 2.04-2.14 (m, 1H) 2.30 (t, J=7.47 Hz, 2H) 2.44-2.61 (m, 4H)2.64-2.69 (m, 1H) 2.72-2.78 (m, 1H) 3.09 (s, 3H) 3.40-3.59 (m, 9H)3.62-3.77 (m, 2H) 3.82-3.89 (m, 1H) 4.12-4.21 (m, 3H) 4.40-4.55 (m, 1H)4.56-4.66 (m, 3H) 5.03-5.12 (m, 2H) 5.55-5.81 (m, 3H) 6.06-6.16 (m, 1H)6.26-6.41 (m, 1H) 6.81 (s, 2H) 7.48-7.57 (m, 2H) 7.78-7.86 (m, 2H).

1.5.1.3 ADL5-D17

General procedure outlined above (section 1.5.1) was employed tosynthesize ADL5-D17. The payload D17 was synthesized using proceduresoutlined in International Application No. PCT/US2019/026992 (see, e.g.,Procedure 2, Procedure 3, and Procedure 4 or 5), which is incorporatedherein by reference.

Step 1:1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6R,7S,10R,E)-2-((E)-1-(3-fluoro-5-morpholinophenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium(12.9 mg, 69.8%). LC/MS (ESI, m/z), 1086.43 [M+H].

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.96-1.07 (m, 12H), 1.27-1.42 (m, 3H),1.46 (d, J=7.15 Hz, 3H), 1.62-1.72 (m, 2H), 1.88 (d, J=1.13 Hz, 3H),1.97-2.06 (m, 1H), 2.04-2.14 (m, 1H), 2.44-2.53 (m, 1H), 2.58-2.69 (m,2H), 3.08 (s, 3H), 3.13-3.19 (m, 4H), 3.38-3.56 (m, 4H), 3.61-3.75 (m,2H), 3.79-3.88 (m, 5H), 3.90-3.98 (m, 1H), 4.07-4.18 (m, 2H), 4.20-4.29(m, 1H), 4.35-4.45 (m, 2H), 4.45-4.54 (m, 1H), 4.61 (s, 2H), 4.86-4.92(m, 2H), 5.15 (d, J=10.67 Hz, 1H), 5.46-5.65 (m, 2H), 6.46-6.67 (m, 4H),7.28-7.37 (m, 2H), 7.37-7.45 (m, 2H), 7.47-7.53 (m, 2H), 7.66-7.71 (m,2H), 7.74-7.84 (m, 4H).

Step 2:1-(4-((R)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6R,7S,10R,E)-2-((E)-1-(3-fluoro-5-morpholinophenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-iumbromide (7.6 mg, 60.5% yield). LC/MS (ESI, m/z), 1057.62 [M+H].

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.95-1.08 (m, 12H), 1.24-1.42 (m, 5H),1.47 (d, J=7.15 Hz, 3H), 1.52-1.71 (m, 6H), 1.89 (d, J=1.13 Hz, 3H),1.95-2.05 (m, 1H), 2.05-2.15 (m, 1H), 2.26-2.34 (m, 2H), 2.46-2.53 (m,1H), 2.58-2.69 (m, 2H), 3.07-3.12 (m, 3H), 3.14-3.19 (m, 4H), 3.39-3.58(m, 6H), 3.61-3.76 (m, 2H), 3.80-3.86 (m, 5H), 4.09-4.18 (m, 3H),4.43-4.51 (m, 1H), 4.62 (s, 2H), 4.87-4.93 (m, 2H), 5.12-5.19 (m, 1H),5.46-5.65 (m, 2H), 6.47-6.66 (m, 4H), 6.80 (s, 2H), 7.53 (d, J=8.66 Hz,2H), 7.82 (d, J=8.66 Hz, 2H).

1.5.1.4 ADL5-D10

General procedure outlined above (section 1.5.1) was employed tosynthesize ADL5-D10. The payload D10 was synthesized using proceduresoutlined in section 1.2.1.

(2S,3S,6S,7R,10R,E)-7-ethoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-methylpiperazine-1-carboxylate (16.6 mg, 0.025 mmol, 38.7% yield).LC/MS (ESI, m/z), 665.7 [M+H]⁺.

¹H-NMR (400 MHz, METHANOL-d): δ ppm 0.76-0.87 (m, 9H), 1.03-1.09 (m,3H), 1.10-1.13 (m, 3H) 1.13-1.19 (m, 1H) 1.20-1.27 (m, 3H), 1.29-1.48(m, 6H) 1.51-1.59 (m, 1H) 1.65-1.71 (m, 3H) 1.73-1.81 (m, 1H) 2.20 (s,3H), 2.27-2.34 (m, 4H) 2.34-2.44 (m, 2H) 2.44-2.53 (m, 1H) 2.53-2.59 (m,1H) 2.76-2.83 (m, 1H) 3.23-3.26 (m, 1H) 3.34-3.52 (m, 7H) 3.67-3.75 (m,1H) 4.86-4.92 (m, 1H) 4.92-4.99 (m, 1H), 5.41-5.51 (m, 1H), 5.60-5.71(m, 1H), 5.72-5.82 (m, 1H), 6.00-6.07 (m, 1H), 6.37-6.48 (m, 1H)

Step 1:1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6S,7R,10R,E)-7-ethoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium(18.6 mg, 0.016 mmol, 87% yield) as a colorless glaze. LC/MS (ESI, m/z),1163.01 [M]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.86-1.03 (m, 16H), 1.20 (s, 3H), 1.25(s, 4H), 1.36 (s, 3H), 1.43-1.72 (m, 10H), 1.81 (s, 3H), 1.84-1.92 (m,1H), 2.06-2.14 (m, 1H), 2.46-2.63 (m, 3H), 2.67-2.72 (m, 1H), 2.88-2.94(m, 1H), 3.03-3.09 (m, 3H), 3.38-3.45 (m, 3H), 3.54 (s, 6H), 3.63-3.79(m, 2H), 3.81-3.88 (m, 1H), 3.92-3.98 (m, 1H), 4.07-4.18 (m, 2H),4.21-4.28 (m, 1H), 4.36-4.44 (m, 2H), 4.47-4.53 (m, 1H), 4.55-4.63 (m,3H), 5.02-5.11 (m, 3H), 5.56-5.66 (m, 1H), 5.75-5.84 (m, 1H), 5.86-5.92(m, 1H), 6.12-6.19 (m, 1H), 6.49-6.61 (m, 1H), 7.29-7.36 (m, 2H),7.38-7.44 (m, 2H), 7.47-7.53 (m, 2H), 7.64-7.71 (m, 2H), 7.80 (dd,J=15.06, 7.91 Hz, 4H).

Step 2:1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6S,7R,10R,E)-7-ethoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium(14.09 mg, 82% yield). LC/MS (ESI, m/z), 1057.62 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.87-1.03 (m, 15H), 1.20 (s, 3H),1.22-1.29 (m, 4H), 1.37 (s, 6H), 1.45-1.72 (m, 14H), 1.81 (s, 3H),1.84-1.95 (m, 1H), 2.05-2.16 (m, 1H), 2.26-2.36 (m, 2H), 2.47-2.62 (m,3H), 2.66-2.74 (m, 1H), 2.86-2.95 (m, 1H), 3.11 (s, 3H), 3.42-3.51 (m,4H), 3.51-3.62 (m, 5H), 3.64-3.78 (m, 2H), 3.81-3.89 (m, 1H), 4.09-4.20(m, 3H), 4.43-4.52 (m, 1H), 4.54-4.59 (m, 1H), 4.55-4.67 (m, 2H),4.60-4.68 (m, 2H), 5.01-5.12 (m, 2H), 5.54-5.65 (m, 1H), 5.75-5.86 (m,1H), 5.86-5.94 (m, 1H), 6.10-6.21 (m, 1H), 6.47-6.62 (m, 1H), 6.81 (s,2H), 7.49-7.60 (m, 2H) 7.76-7.89 (m, 2H).

1.5.1.5 ADL5-D15

General procedure outlined above (section 1.5.1) was employed tosynthesize ADL5-D15. The payload D15 was synthesized using proceduresoutlined in International Application No. PCT/US2019/026992, which isincorporated herein by reference.

Step 1:1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6R,7S,10R,E)-2-((E)-1-(1-(1-acetylpiperidin-4-yl)-4-fluoro-1H-benzo[d][1,2,3]triazol-6-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium(100 mg, 0.086 mmol, 57.3% yield). LC/MS (ESI, m/z), 1167.76 [M+H].

Step 2:4-((((2S,3S,6R,7S,10R,E)-2-((E)-1-(1-(1-acetylpiperidin-4-yl)-4-fluoro-1H-benzo[d][1,2,3]triazol-6-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-1-methylpiperazin-1-ium(32 mg, 0.028 mmol, 32.8% yield). LC/MS (ESI, m/z), 1138.64 [M+H].

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.99 (s, 10H), 1.26-1.40 (m, 5H),1.42-1.49 (m, 3H), 1.67 (br s, 6H), 1.96 (d, J=1.00 Hz, 3H), 2.00-2.13(m, 2H), 2.16-2.42 (m, 7H), 2.44-2.55 (m, 1H), 2.59-2.76 (m, 2H),2.98-3.26 (m, 4H), 3.38-3.59 (m, 7H), 3.60-3.76 (m, 2H), 3.78-3.90 (m,1H), 4.13 (br d, J=7.15 Hz, 3H), 4.41-4.52 (m, 1H), 4.53-4.71 (m, 3H),4.89-4.97 (m, 1H), 5.10-5.27 (m, 2H), 5.48-5.67 (m, 2H), 6.79 (s, 2H)7.07-7.17 (m, 1H), 7.48-7.61 (m, 3H), 7.78-7.84 (m, 2H).

1.5.1.5 ADL5-D32

General procedure outlined above (section 1.5.1) was employed tosynthesize ADL5-D32. The payload D15 was synthesized using proceduresoutlined in International Application No. PCT/US2019/026992, which isincorporated herein by reference.

Step 1:1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6R,7S,10R,E)-2-((E)-1-(5-chloropyridin-3-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium(15 mg, 0.015 mmol, 69.6% yield) LC/MS (ESI, m/z), 1018.4 [M]⁺

1H NMR (400 MHz, METHANOL-d4) δ ppm 0.99 (s, 14H) 1.29-1.41 (m, 3H)1.44-1.49 (m, 3H) 1.61-1.71 (m, 2H) 1.92 (d, J=1.25 Hz, 3H), 1.95-2.13(m, 2H) 2.45-2.53 (m, 1H) 2.60-2.70 (m, 2H) 3.04-3.09 (m, 3H) 3.37 (s,3H) 3.39-3.47 (m, 1H) 3.47-3.54 (m, 2H) 3.61-3.75, (m, 2H) 3.78-3.87 (m,1H) 3.93-3.99 (m, 1H) 4.08-4.18 (m, 2H) 4.22-4.29 (m, 1H) 4.39-4.45 (m,2H) 4.45-4.53 (m, 1H) 4.56-4.63 (m, 3H) 5.17-5.23 (m, 1H) 5.48-5.64 (m,2H) 6.57-6.64 (m, 1H) 7.28-7.36 (m, 2H) 7.37-7.45 (m, 2H) 7.47-7.54 (m,2H) 7.64-7.70 (m, 2H) 7.75-7.84 (m, 5H) 8.41-8.48 (m, 2H)

Step 2:4-((((2S,3S,6R,7S,10R,E)-2-((E)-1-(5-chloropyridin-3-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-1-methylpiperazin-1-ium(6.2 mg, 42.5 yield). LC/MS (ESI, m/z), 990.34 [M+H].

1H NMR (400 MHz, METHANOL-d4) δ ppm 0.97-1.09 (m, 12H) 1.24-1.41 (m, 5H)1.47 (d, J=7.15 Hz, 3H) 1.55-1.71 (m, 6H) 1.92 (d, J=1.13 Hz, 3H)1.98-2.15 (m, 2H) 2.27-2.36 (m, 2H) 2.46-2.53 (m, 1H) 2.60-2.71 (m, 2H)3.06-3.13 (m, 3H) 3.35-3.38 (m, 1H) 3.41-3.58 (m, 6H) 3.62-3.76 (m, 2H)3.80-3.88 (m, 1H) 4.08-4.19 (m, 3H) 4.43-4.52 (m, 1H) 4.57-4.66 (m, 2H)5.16-5.24 (m, 1H) 5.49-5.68, (m, 2H) 6.62 (s, 1H) 6.81 (s, 2H) 7.49-7.57(m, 2H) 7.76-7.87 (m, 3H) 8.39-8.49 (m, 2H)

1.5.1.5 ADL5-D30

General procedure outlined above (section 1.5.1) was employed tosynthesize ADL5-D30. The payload D30 was synthesized using proceduresoutlined in International Application No. PCT/US2019/026992, which isincorporated herein by reference.

Step 1:1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6R,7S,10R,E)-2-((E)-1-(3-(dimethylamino)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium(16 mg, 0.016 mmol, 51.4% yield) LC/MS (ESI, m/z), 1027.50 [M+H]⁺

1H NMR (400 MHz, METHANOL-d₄) δ ppm 0.95-1.07 (m, 13H) 1.31-1.42 (m, 2H)1.44-1.50 (m, 3H) 1.63-1.71 (m, 2H) 1.84-1.92 (m, 3H) 1.98-2.14 (m, 2H)2.47-2.56 (m, 1H) 2.60-2.67 (m, 2H) 2.93 (s, 6H) 3.03-3.09 (m, 3H) 3.37(s, 4H) 3.47-3.56 (m, 2H) 3.63-3.75 (m, 2H) 3.79-3.88 (m, 1H) 3.92-3.98(m, 1H) 4.08-4.19 (m, 2H) 4.21-4.27 (m, 1H) 4.37-4.44 (m, 2H) 4.45-4.54(m, 1H) 4.57-4.62 (m, 2H) 4.91-4.94 (m, 2H) 5.15-5.20 (m, 1H) 5.47-5.55(m, 1H) 5.56-5.65 (m, 1H) 6.58 (s, 1H) 6.67 (d, J=1.51 Hz, 3H) 7.15-7.24(m, 1H), 7.28-7.36 (m, 2H) 7.38-7.45 (m, 2H) 7.47-7.54 (m, 2H) 7.64-7.73(m, 2H) 7.75-7.85 (m, 4H)

Step 2:4-((((2S,3S,6R,7S,10R,E)-2-((E)-1-(5-chloropyridin-3-yl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-1-methylpiperazin-1-ium(6.2 mg, 42.5 yield). LC/MS (ESI, m/z), 990.34 [M+H].

1H NMR (400 MHz, METHANOL-d4) δ ppm 0.88 (d, J=6.65 Hz, 12H) 1.16-1.30(m, 4H) 1.32-1.38 (m, 3H) 1.43-1.58 (m, 6H) 1.75-1.80, (m, 3H) 1.84-1.92(m, 1H) 1.93-2.02 (m, 1H) 2.14-2.21 (m, 2H) 2.35-2.42 (m, 1H) 2.48-2.57(m, 2H) 2.82 (s, 6H) 2.94-3.02 (m, 3H), 3.27-3.32 (m, 1H) 3.35-3.45 (m,4H) 3.50-3.64 (m, 2H) 3.68-3.76 (m, 1H) 3.97-4.07 (m, 3H) 4.31-4.40 (m,1H) 4.47-4.53 (m, 2H) 5.03-5.09 (m, 1H) 5.35-5.44 (m, 1H) 5.45-5.55 (m,1H) 6.42-6.50 (m, 1H) 6.51-6.62 (m, 3H) 6.69 (s, 2H) 7.03-7.12 (m, 1H)7.38-7.44 (m, 2H) 7.66-7.75 (m, 2H) 8.42-8.47 (m, 1H).

1.5.1.5 ADL5-D27

General procedure outlined above (section 1.5.1) was employed tosynthesize ADL5-D27. The payload D27 was synthesized using proceduresoutlined in International Application No. PCT/US2019/026992, which isincorporated herein by reference.

Step 1:1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6R,7S,10R,E)-2-((E)-1-(3-fluoro-5-(4-((1-methyl-1H-pyrazol-4-yl)sulfonyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium(25.2 mg, 0.020 mmol, 83% yield). LC/MS (ESI, m/z), 1230.54 [M+H]⁺

Step 2:1-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6R,7S,10R,E)-2-((E)-1-(3-fluoro-5-(4-((1-methyl-1H-pyrazol-4-yl)sulfonyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium(5.2 mg, 4.33 μmol, 21.13% yield). LC/MS (ESI, m/z), 1201.68 [M+H]⁺

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 0.87 (d, J=6.78 Hz, 12H) 1.13-1.29(m, 4H) 1.31-1.38 (m, 3H) 1.43-1.59 (m, 6H) 1.71-1.79, (m, 3H) 1.83-2.02(m, 2H) 2.14-2.21 (m, 2H) 2.34-2.41 (m, 1H) 2.56 (s, 8H) 2.94-3.01 (m,3H) 3.12-3.18 (m, 8H) 3.28-3.45 (m, 5H), 3.48-3.63 (m, 2H) 3.70 (s, 4H)3.97-4.07 (m, 3H) 4.29-4.41 (m, 1H) 4.49 (s, 2H) 4.98-5.06 (m, 1H)5.34-5.54 (m, 2H) 6.37-6.45 (m, 2H) 6.51 (s, 2H) 6.69 (s, 2H) 7.37-7.43(m, 2H) 7.63-7.73 (m, 4H) 1.5.1.6 ADL10-D1

(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (0.012 g, 0.019 mmol), 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (0.012 g, 0.038 mmol),and DCM (0.188 ml, 0.019 mmol) were combined and stirred overnight. Themixture was directly loaded onto a silica gel column and chromatographedto afford(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)piperazine-1-carboxylate(8.4 mg, 10.12 μmol, 53.7% yield). LC/MS [M+Na] 853.5.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.89 (s, 9H) 1.21-1.39 (m, 9H)1.41-1.56 (m, 5H) 1.57-1.71 (m, 6H) 1.69-1.71 (m, 1H) 1.79, (d, J=0.88Hz, 3H) 1.82-1.90 (m, 1H) 2.34-2.43 (m, 1H) 2.41-2.41 (m, 1H) 2.46-2.63(m, 3H) 2.65-2.68 (m, 2H) 2.68 (s, 3H) 2.86-2.92 (m, 1H) 3.33 (s, 3H)3.51 (br d, J=7.03 Hz, 6H) 3.54-3.60 (m, 5H) 3.77-3.88 (m, 1H) 5.06 (s,2H) 5.53-5.63 (m, 1H) 5.70-5.80 (m, 1H), 5.83-5.94 (m, 1H) 6.08-6.19 (m,1H) 6.47-6.59 (m, 1H) 6.80 (s, 2H).

1.6 Preparation of ADL12-D1, ADL14-D1, and ADL15-D1 1.6.1Overview—General Procedure 1

For ADL12-D1—Step 1: To a stirred solution of1-(6-hydroxyhexyl)-1H-pyrrole-2,5-dione (0.200 g, 1.014 mmol) in DCM(20.28 mL, 1.014 mmol) was added PDC (3.81 g, 10.14 mmol) and aceticacid (0.1 mL). The reaction mixture was stirred for 3 hours at RT. Thereaction mixture was then filtered through a silica pad and concentratedto dryness to afford 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanal (40mg, 0.205 mmol, 20.21% yield).

For ADL14-D1 and ADL15-D1—Step 1: To a stirred solution of maleimidealcohol (1.0 equiv.) in DCM (0.05 M) was added pre-activated powderedMS-4A 2.6 mg per 1 mg of alcohol, N-methylmorpholine-N-oxide (1.2equiv.), and TPAP (0.1 equiv.) in that order. The reaction mixture wasstirred for 60 min at RT. The reaction mixture was then filtered througha silica gel pad and concentrated to dryness. Unpurified product wastaken forward to the next step (Step 2).

Step 2:(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylpiperazine-1-carboxylate (1.0 equiv.),2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetaldehyde (2.0-3.0 equiv.),DCM (0.1 M), sodium triacetoxyborohydride (3.0 equiv.) were mixed andstirred for 10 min. The reaction mixture was then loaded directly onto asilica gel column and chromatographed to afford the desiredlinker-payload.

1.6.1.1 ADL12-D1

Linker-Payload (ADL12-D1): General procedure outlined in section 1.6.1was employed to synthesize(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)piperazine-1-carboxylate(41 mg, 0.050 mmol, 80% yield). LC/MS [M+]⁺816.75.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.86-0.98 (m, 9H) 1.20-1.23 (m, 3H)1.23-1.40 (m, 8H) 1.41-1.70 (m, 12H) 1.79 (d, J=0.63 Hz, 3H) 1.83-1.91(m, 1H) 2.33-2.40 (m, 2H) 2.43 (br t, J=4.77 Hz, 4H) 2.48-2.64 (m, 3H)2.65-2.69 (m, 1H) 2.87-2.92 (m, 1H) 3.32 (s, 3H) 3.41-3.60 (m, 7H)3.79-3.87 (m, 1H) 5.04 (dd, J=19.39, 10.23 Hz, 2H) 5.57 (dd, J=15.18,9.79 Hz, 1H) 5.69-5.79 (m, 1H) 5.87 (d, J=15.31 Hz, 1H) 6.11-6.17 (m,1H) 6.49-6.58 (m, 1H) 6.80 (s, 2H).

1.6.1.X ADL12-D28

Linker-Payload (ADL12-D28): General procedure outlined in section 1.6.1was employed to synthesize(2S,3S,6R,7S,10R,E)-2-((E)-1-(3-fluoro-5-(4-((1-methyl-1H-pyrazol-4-yl)sulfonyl)piperazin-1-yl)phenyl)prop-1-en-2-yl)-10-hydroxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)piperazine-1-carboxylate(2.3 mg, 30.7%). LC/MS [M+]⁺ 897.32

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 0.85-0.93 (m, 6H) 1.16-1.34 (m, 6H)1.37-1.58 (m, 6H) 1.72-1.77 (m, 3H) 1.80-1.88 (m, 1H), 2.23-2.41 (m, 7H)2.47-2.57 (m, 2H) 3.08-3.19 (m, 9H) 3.33-3.42 (m, 5H) 3.70 (s, 4H)4.67-4.72 (m, 1H) 4.98-5.07 (m, 1H) 5.33-5.50 (m, 2H) 6.36-6.43 (m, 2H)6.44-6.53 (m, 2H) 6.70 (s, 2H) 7.62-7.69 (m, 2H)

1.6.1.X ADL12-D35

Linker-Payload (ADL12-D35): General procedure outlined in section 1.6.1was employed to synthesize(2S,3S,6R,7S,10R,E)-10-hydroxy-3,7-dimethyl-12-oxo-2-((E)-1-(3-(pyrrolidin-1-ylsulfonyl)phenyl)prop-1-en-2-yl)oxacyclododec-4-en-6-yl4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)piperazine-1-carboxylate(4.0 mg, 5.11 μmol, 28.8% yield). LC/MS [M+H]⁺ 783.5

1H NMR (400 MHz, METHANOL-d4) δ ppm 0.91 (dd, J=6.78, 2.51 Hz, 6H)1.17-1.33 (m, 6H) 1.38-1.59 (m, 6H) 1.65 (s, 4H) 1.80 (s, 3H), 1.82-1.91(m, 1H) 2.23-2.41 (m, 7H) 2.49-2.61 (m, 2H) 3.14 (s, 4H) 3.38 (br d,J=7.15 Hz, 6H) 3.67-3.76 (m, 1H) 4.68-4.73 (m, 1H) 5.03-5.11 (m, 1H)5.34-5.52 (m, 2H) 6.55-6.62 (m, 1H) 6.70 (s, 2H) 7.46-7.54 (m, 2H)7.58-7.65 (m, 2H)

1.6.1.X ADL12-D22 (R)

Linker-Payload (ADL12-D28): General procedure outlined in section 1.6.1was employed to synthesize3-((R)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoicacid (3.2 mg, 3.60 μmol, 15.39% yield). LC/MS [M+]⁺ 888.5

1.6.1.X ADL12-D22 (S)

Linker-Payload (ADL12-D28): General procedure outlined in section 1.6.1was employed to synthesize3-((S)-1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoicacid (3.6 mg, 3.60 μmol, 28.7% yield). LC/MS [M+]⁺ 888.56

1.6.1.2 ADL14-D1

Linker-Payload (ADL14-D1): General procedure outlined in section 1.6.1was employed to synthesize(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)piperazine-1-carboxylate(8 mg, 10.53 μmol, 33.5% yield). LC/MS [M+H] 760.3.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.85-1.01 (m, 9H), 1.17-1.33 (m, 5H),1.36 (s, 3H), 1.41-1.71 (m, 7H), 1.80 (s, 3H), 1.83-1.95 (m, 1H), 2.48(br s, 10H), 2.89-2.98 (m, 1H), 3.40-3.57 (m, 5H), 3.61-3.72 (m, 2H),3.78-3.91 (m, 1H), 4.98-5.14 (m, 2H), 5.50-5.52 (m, 1H), 5.52-5.65 (m,1H), 5.69-5.82 (m, 1H), 5.86-5.96 (m, 1H), 6.09-6.21 (m, 1H), 6.47-6.60(m, 1H), 6.79-6.90 (m, 2H).

1.6.1.3 ADL15-D1

Linker-Payload (ADL15-D1): General procedure outlined in section 1.6.1was employed to synthesize(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)piperazine-1-carboxylate(7 mg, 8.71 μmol, 23.10% yield). LC/MS [M=+H] 804.1.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.85-1.02 (m, 9H), 1.18-1.32 (m, 4H),1.36 (s, 3H), 1.40-1.72 (m, 7H), 1.78-1.83 (m, 3H), 1.85-1.93 (m, 1H),2.42-2.65 (m, 9H), 2.67-2.73 (m, 1H), 2.89-2.96 (m, 1H), 3.37 (s, 5H),3.41-3.58 (m, 5H), 3.59-3.67 (m, 4H), 3.68-3.75 (m, 2H), 3.81-3.89 (m,1H), 4.94-5.15 (m, 2H), 5.61 (br d, J=10.04 Hz, 1H), 5.69-5.82 (m, 1H),5.89 (br d, J=15.18 Hz, 1H), 6.16 (br d, J=10.92 Hz, 1H), 6.56 (br s,1H), 6.77-6.91 (m, 2H).

1.7 ADL12-D2

(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)piperazine-1-carboxylate(26 mg, 0.032 mmol), acetonitrile (319 μL, 0.032 mmol), iodomethane(19.92 μL, 0.319 mmol) were combined and stirred overnight. The reactionmix was concentrated to dryness and chromatographed (0-30% MeOH in DCM)to afford1-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-methylpiperazin-1-ium(14 mg, 0.017 mmol, 52.9% yield). LC/MS [M+] 831.6.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.86-0.98 (m, 9H) 1.26 (s, 4H) 1.36 (s,3H) 1.40-1.58 (m, 9H) 1.60-1.71 (m, 4H) 1.80 (d, J=0.88 Hz, 4H)1.85-1.91 (m, 1H) 1.87-1.88 (m, 1H) 2.48-2.63 (m, 3H) 2.65-2.72 (m, 1H)2.88-2.94 (m, 1H) 3.18 (s, 3H) 3.34-3.41 (m, 3H) 3.53 (s, 10H) 3.73-3.90(m, 3H) 3.90-4.06 (m, 2H) 5.02-5.11 (m, 2H) 5.56-5.66 (m, 1H) 5.70-5.81(m, 1H) 5.84-5.94 (m, 1H) 6.11-6.20 (m, 1H) 6.50-6.60 (m, 1H) 6.83 (s,2H).

1.8 ADL15-D2

(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)piperazine-1-carboxylate(6 mg, 7.463 μmol), iodomethane (0.467 μL, 7.463 μmol), and acetonitrile(0.390 μL, 7.463 μmol) were combined and stirred at RT. The reactionmixture was concentrated to dryness to afford(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethoxy)ethyl)-4-methyl-414-piperazine-1-carboxylate(6 mg, 7.33 μmol, 98% yield). LC/MS [M+]⁺ 819.28.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.74-0.88 (m, 9H) 1.18 (br d, J=4.27Hz, 5H) 1.23-1.27 (m, 3H) 1.28-1.49 (m, 5H) 1.51-1.61 (m, 2H) 1.66-1.71(m, 3H) 1.73-1.80 (m, 1H) 2.36-2.53 (m, 3H) 2.55-2.60 (m, 1H) 2.78-2.83(m, 1H) 3.10-3.14 (m, 3H) 3.23-3.25 (m, 3H) 3.32-3.51 (m, 5H) 3.52-3.67(m, 6H) 3.67-3.87 (m, 7H) 4.93-5.01 (m, 2H) 5.44-5.55 (m, 1H) 5.61-5.70(m, 1H) 6.00-6.07 (m, 1H) 6.38-6.48 (m, 1H) 6.75 (s, 2H).

1.9 ADL5-D25

Step 1: D1 (40 mg, 0.063 mmol) was dissolved in N,N-dimethylformamide(730 μl, 9.422 mmol) and methyl-bromobutarate (23.69 μL, 0.188 mmol) wasadded followed by triethylamine (35.0 μL, 0.251 mmol). The solution wasstirred at RT for 2 days. The solvent was evaporated. Purification bycolumn chromatography afforded(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(4-methoxy-4-oxobutyl)piperazine-1-carboxylate (51 mg, 0.069 mmol,˜100% yield) as a colorless oil. LC/MS (ESI, m/z), 737.4 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.76-0.86 (m, 9H), 1.12 (s, 3H),1.14-1.19 (m, 1H), 1.24 (s, 3H), 1.34-1.50 (m, 5H), 1.52-1.60 (m, 1H),1.69 (s, 6H), 2.27 (s, 8H), 2.37-2.49 (m, 3H), 2.54-2.61 (m, 1H),2.77-2.83 (m, 1H), 3.35-3.47 (m, 5H), 3.56 (s, 3H), 3.67-3.76 (m, 1H),4.46 (s, 3H), 4.89-5.00 (m, 3H), 5.42-5.52 (m, 1H), 5.57-5.70 (m, 1H),5.73-5.81 (m, 1H), 5.98-6.08 (m, 1H), 6.43 (dd, J=15.25, 10.98 Hz, 1H).

Step 2: Fmoc-ValAla-PAB bromide (88 mg, 0.152 mmol) and(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(4-methoxy-4-oxobutyl)piperazine-1-carboxylate (51 mg, 0.069 mmol)were suspended in N,N-dimethylformamide (670 μL, 8.651 mmol). Thereaction was stirred at RT overnight. The solvent was evaporated.Purification by column chromatography afforded1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-(4-methoxy-4-oxobutyl)piperazin-1-ium(42 mg, 0.034 mmol, 49.1% yield) as a waxy solid. LC/MS (ESI, m/z),1235.4 [M]⁺.

Step 3:1-(4-((S)-2-((S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)-1-(4-methoxy-4-oxobutyl)piperazin-1-ium(42 mg, 0.034 mmol) was dissolved in a 5:2:1 ratio of THF (600 μL, 7.322mmol), MeOH (250 μL, 6.179 mmol), water (125 μL, 6.939 mmol), andlithium hydroxide (1.221 mg, 0.051 mmol) was added. The mixture wasstirred at RT for 1 hour. The mixture was quenched with acetic acid(5.84 μL, 0.102 mmol) and the solvent was evaporated and purified by RPHPLC to afford1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl)-1-(3-carboxypropyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-1-iumas a waxy solid (18.4 mg, 0.018 mmol, 54.2% yield). LC/MS (ESI, m/z),998.5 [M]⁺.

Step 4:1-(4-((S)-2-((S)-2-amino-3-methylbutanamido)propanamido)benzyl)-1-(3-carboxypropyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-1-ium(18.4 mg, 0.018 mmol) was dissolved in N,N-dimethylformamide (356 μL,4.603 mmol) and 2,5-dioxopyrrolidin-1-yl6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (6.81 mg, 0.022 mmol)was added followed by Hunig's base (8.04 μL, 0.046 mmol). The reactionsolution was stirred at RT for 30 min. The solvent was evaporated andthe crude residue was purified by RP HPLC to afford(1-(3-carboxypropyl)-1-(4-((S)-2-((R)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-1-ium(8.8 mg, 7.38 μmol, 40.1% yield) as a white lyophilized solid. LC/MS(ESI, m/z), 1191.5 [M]⁺.

¹H-NMR (400 MHz, DMSO-d6): δ ppm 0.68-0.81 (m, 18H), 0.98-1.07 (m, 4H),1.07-1.14 (m, 3H), 1.16 (br d, J=3.89 Hz, 5H), 1.22-1.33 (m, 8H),1.36-1.47 (m, 7H), 1.62 (s, 3H) 1.66-1.73 (m, 1H), 1.83-1.92 (m, 5H),1.98-2.13 (m, 2H), 2.26 (dt, J=3.54, 1.80 Hz, 1H), 2.27-2.33 (m, 1H),2.47-2.51 (m, 2H), 2.58-2.62 (m, 1H), 2.66-2.72 (m, 1H), 3.15 (s, 3H)3.45-3.58 (m, 3H), 3.60-3.68 (m, 2H) 3.77-3.86 (m, 2H), 4.06-4.13 (m,1H), 4.27-4.37 (m, 2H), 4.46-4.54 (m, 3H), 4.72-4.78 (m, 1H), 4.80-4.87(m, 2H), 5.32-5.48 (m, 1H), 5.53-5.67 (m, 1H), 5.73-5.86 (m, 1H),5.93-6.05 (m, 1H), 6.16-6.26 (m, 1H), 6.20 (s, 1H), 6.23-6.41 (m, 1H),6.27-6.38 (m, 1H), 6.93 (s, 2H), 6.98-7.06 (m, 2H), 7.43-7.50 (m, 2H),7.62-7.69 (m, 2H), 7.74-7.80 (m, 1H), 8.22-8.27 (m, 1H), 8.44-8.49 (m,4H), 10.15-10.22 (m, 1H).

1.10 ADL12-D20 & ADL12-D21

General procedure 1 outlined in section 1.2.1 was employed to synthesizeD20.

(2S,3S,6S,7R,10R,E)-7-methoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-ylmethyl(2-(methylamino)ethyl)carbamate (43 mg, 0.044 mmol, 90% yield) asa colorless oil. LC/MS (ESI, m/z), 981.4 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.62-0.73 (m, 18H), 0.82-0.93 (m, 9H),0.95-1.06 (m, 27H), 1.24 (br s, 4H), 1.45 (br s, 10H), 1.78 (br s, 3H),1.92-2.00 (m, 1H), 2.46 (br s, 4H), 2.51-2.67 (m, 3H), 2.76-2.84 (m,2H), 2.86-2.93 (m, 1H), 2.93-3.02 (m, 3H), 3.34-3.38 (m, 3H), 3.43-3.49(m, 2H), 3.72-3.82 (m, 1H), 3.93-4.04 (m, 1H), 4.94-5.07 (m, 2H),5.53-5.64 (m, 1H), 5.72-5.89 (m, 2H), 6.07-6.21 (m, 1H), 6.44-6.61 (m,1H).

D20

(2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-ylmethyl(2-(methylamino)ethyl)carbamate (16.4 mg, 0.026 mmol, 58.6% yield)as a colorless oil. LC/MS (ESI, m/z), 639.7 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.92 (br d, J=6.53 Hz, 9H), 1.25 (br s,4H), 1.36 (s, 3H), 1.41-1.74 (m, 6H), 1.45-1.59 (m, 1H), 1.64-1.72 (m,1H), 1.80 (s, 3H), 1.85-1.94 (m, 1H), 2.42 (s, 3H), 2.50-2.63 (m, 3H),2.65-2.73 (m, 1H), 2.73-2.80 (m, 2H), 2.88-3.01 (m, 4H), 3.35-3.38 (m,3H), 3.35-3.40 (m, 3H), 3.41-3.48 (m, 2H), 3.52-3.57 (m, 1H), 3.78-3.89(m, 1H), 5.00-5.13 (m, 2H), 5.53-5.64 (m, 1H), 5.71-5.82 (m, 1H),5.84-5.94 (m, 1H), 6.12-6.19 (m, 1H), 6.49-6.61 (m, 1H).

ADL12-D20

General procedure outlined in section 1.6.1 was employed to synthesizeALD12-D20 and afforded (13.0 mg, 0.016 mmol, 67.7%) as a colorless oil.LC/MS (ESI, m/z), 818.3 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.91 (br s, 9H), 1.21-1.31 (m, 4H),1.36 (br s, 7H), 1.46-1.73 (m, 11H), 1.80 (s, 3H), 1.84-1.93 (m, 1H),2.50-2.72 (m, 7H), 2.75-2.85 (m, 1H), 2.87-3.09 (m, 7H), 3.48-3.68 (m,6H) 3.80-3.89 (m, 1H), 5.02-5.13 (m, 2H), 5.55-5.66 (m, 1H), 5.71-5.82(m, 1H), 5.85-5.94 (m, 1H), 6.11-6.22 (m, 1H), 6.49-6.61 (m, 1H), 6.83(br d, J=1.13 Hz, 2H).

ADL12-D21

((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl(2-((6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexyl)(methyl)amino)ethyl)(methyl)carbamate(7 mg, 8.557 μmol) was dissolved in N,N-dimethylformamide (99 μl, 1.284mmol) and methyl iodide (2.68 μL, 0.043 mmol) was added. The solutionwas stirred at rt o/n. The solvent was evaporated. Purification bycolumn chromatography afforded6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-(((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)(methyl)amino)ethyl)-N,N-dimethylhexan-1-aminium(7 mg, 8.40 μmol, 98% yield). LC/MS (ESI, m/z), 832.7 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.75-0.87 (m, 9H), 1.14 (s, 4H), 1.25(s, 3H), 1.27-1.45 (m, 9H), 1.48-1.61 (m, 4H), 1.69 (s, 4H), 1.72-1.79(m, 2H), 2.35-2.53 (m, 3H), 2.53-2.60 (m, 1H), 2.75-2.83 (m, 1H),2.85-2.95 (m, 3H), 3.06 (br d, J=10.42 Hz, 6H), 3.15-3.20 (m, 2H), 3.25(br d, J=2.38 Hz, 3H), 3.42 (br d, J=6.90 Hz, 5H), 3.53-3.69 (m, 2H),3.69-3.78 (m, 1H), 4.88-5.01 (m, 2H), 5.44-5.56 (m, 1H), 5.59-5.72 (m,1H), 5.73-5.83 (m, 1H), 5.99-6.10 (m, 1H), 6.40 (s, 1H), 6.72 (s, 2H).1.11 ADL12-D22

General procedure 1 outlined in section 1.2.1 was employed to synthesizeD22.

3-(4-((((2S,3S,6S,7R,10R,E)-7-methoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoicacid (48 mg, 0.046 mmol, 34.4% yield) as a colorless oil. LC/MS (ESI,m/z), 1051.4 [M+H]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.62-0.70 (m, 18H), 0.82-0.94 (m, 9H),1.01 (tt, J=7.92, 3.18 Hz, 27H), 1.22-1.28 (m, 4H), 1.45 (s, 3H),1.47-1.66 (m, 7H), 1.76-1.87 (m, 5H), 1.92-1.99 (m, 1H), 2.36-2.47 (m,3H), 2.50-2.68 (m, 3H), 2.86-2.93 (m, 1H), 2.98-3.08 (m, 2H), 3.10-3.18(m, 1H), 3.18-3.26 (m, 2H), 3.27-3.31 (m, 1H), 3.34-3.36 (m, 3H),3.36-3.37 (m, 1H) 3.72-3.81 (m, 1H), 3.95-4.19 (m, 3H), 4.93-5.00 (m,1H), 5.01-5.07 (m, 1H), 5.54-5.66 (m, 1H) 5.72-5.81 (m, 1H) 5.81-5.90(m, 1H), 6.12-6.19 (m, 1H), 6.47-6.58 (m, 1H).

D22

N-ethyl-N-isopropylpropan-3-(4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoicacid (23.5 mg, 0.031 mmol, 68.2% yield). LC/MS (ESI, m/z), 709.5 [M+H]⁺.

¹H-NMR (400 MHz, DMSO-d6): δ ppm 0.71-0.77 (m, 9H), 1.03 (s, 5H),1.14-1.20 (m, 6H), 1.23-1.33 (m, 5H), 1.36-1.48 (m, 5H), 1.63 (s, 3H),1.67-1.75 (m, 1H), 2.13-2.24 (m, 4H), 2.58-2.64 (m, 1H), 2.68-2.81 (m,2H), 3.20-3.20 (m, 3H), 3.61-3.77 (m, 4H), 4.28-4.39 (m, 1H), 4.44-4.53(m, 1H), 4.79-4.88 (m, 2H), 5.28-5.43 (m, 1H), 5.52-5.66 (m, 1H),5.73-5.85 (m, 1H), 5.94-6.04 (m, 1H), 6.27-6.41 (m, 1H), 6.43-6.53 (m,2H), 8.19-8.29 (m, 1H).

ADL12-D22

General procedure outlined in section 1.6.1 was employed to synthesizeADL12-D22 (3.6 mg, 4.05 μmol, 34.4% yield). LC/MS (ESI, m/z), 888.7[M+H]⁺.

¹H-NMR (400 MHz, MeOH-d4): δ ppm 0.77-0.89 (m, 9H), 1.08-1.31 (m, 13H),1.35-1.60 (m, 12H), 1.69 (s, 3H), 1.73-1.83 (m, 1H), 1.85-1.97 (m, 1H),2.15-2.27 (m, 1H), 2.27-2.83 (m, 11H), 2.84-2.98 (m, 1H), 3.39 (s, 7H),3.67-3.77 (m, 1H), 4.89-5.01 (m, 2H), 5.40-5.53 (m, 1H), 5.59-5.69 (m,1H), 5.72-5.83 (m, 1H), 5.99-6.11 (m, 1H), 6.37-6.49 (m, 1H), 6.69 (s,2H).

ADL1-D22

General procedure 1 outlined in section 1.2.1 was employed to synthesizeADL1-D22 (4.7 mg, 3.59 μmol, 38.6% yield) as a white lyophilized solid.LC/MS (ESI, m/z), 1307.6 [M+H]⁺.

¹H NMR (400 MHz, DMSO-d6): δ ppm 0.68-0.79 (m, 16H), 1.03 (s, 3H), 1.16(s, 5H), 1.23-1.44 (m, 13H), 1.48-1.55 (m, 1H), 1.62 (s, 4H), 1.68-1.74(m, 1H), 1.86-1.93 (m, 1H), 2.02 (s, 4H), 2.24-2.37 (m, 3H), 2.46-2.51(m, 2H), 2.66-2.73 (m, 1H), 2.79-2.99 (m, 4H), 3.15 (s, 3H), 3.21-3.22(m, 2H), 3.30 (s, 4H), 3.61-3.67 (m, 1H), 3.73-3.88 (m, 3H), 4.01-4.08(m, 1H), 4.09-4.16 (m, 1H), 4.29-4.38 (m, 2H), 4.43-4.51 (m, 1H),4.73-4.78 (m, 1H), 4.79-4.88 (m, 2H), 4.91-4.98 (m, 2H), 5.29-5.43 (m,3H), 5.54-5.67 (m, 1H), 5.74-5.83 (m, 1H), 5.95-6.07 (m, 1H), 6.28-6.40(m, 1H), 6.54-6.62 (m, 1H), 6.93 (s, 2H), 7.17-7.26 (m, 2H), 7.44-7.57(m, 2H), 7.66-7.76 (m, 1H), 7.95-8.03 (m, 1H), 8.42-8.49 (m, 1H),9.89-9.96 (m, 1H).

ADL1-D23

4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (16.76 mg, 0.023 mmol) and3-((R)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoicacid (16.1 mg, 0.023 mmol) were dissolved in DMF (229 μl, 2.953 mmol)and Hunig's Base (11.90 μl, 0.068 mmol) was added. The reaction mixturewas stirred overnight at rt. The solvent was then evaporated andsubjected to reverse-phase HPLC purification to afford3-((R)-1-(((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoicacid (3.9 mg, 2.98 μmol, 13.13% yield). LC/MS (ESI, m/z), 1308.6 [M+H]⁺.

ADL1-024 (S)

3-((S)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoicacid (10 mg, 0.014 mmol), N,N-dimethylformamide (141 μl, 0.014 mmol),Hunig's base (5 μl, 0.028 mmol),4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl) carbonate (20.8 mg, 0.028 mmol) were combined andstirred for 2 hours. chromatograoged (MeOH/CH₂Cl₂) to afford3-((S)-1-(((4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl)oxy)carbonyl)-4-((((2S,3S,6S,7R,10R,E)-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-7-methoxy-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl)oxy)carbonyl)piperazin-2-yl)propanoicacid (5 mg, 3.82 μmol, 27.1% yield). LC/MS (ESI, m/z), 1308.4 [M+H]⁺.

Example 2

Exemplary spliceosome modulator payloads used in the preparation of ADCswere profiled. Payloads were evaluated for binding to the SF3b complex,in vitro splicing activity, and ability to inhibit cell growth.

2.1 SF3B1 Binding/Scintillation Proximity Assay (SPA)

A scintillation proximity assay was performed to measure the bindingaffinity of compounds (“payloads”) to the SF3b complex. Batchimmobilization of anti-SF3B1 antibody (MBL) to anti-mouse PVT SPAscintillation beads (PerkinElmer) was prepared as follows: for every 2.5mg of nuclear extracts, 5 μg of anti-SF3B1 antibody and 1.5 mg of beadswere mixed in 150 μL PBS. The antibody-bead mixture was incubated for 30min at RT and centrifuged at 18,000 g for 5 min. 150 μL PBS was used toresuspend every 1.5 mg antibody-bead mixture. The beads were suspendedand added to the prepared nuclear extracts. The slurry was incubated for2 hours at 4° C. with gentle mixing. The beads were then collected bycentrifuging at 18,000 g for 5 min, and washed twice with PBS +0.1%Triton X-100. After a final centrifugation step, every 1.5 mg of beadswas suspended with 150 μL of PBS. SF3b complexes were tested for[³H]-labeled pladienolide B probe binding ([³H]-PB), synthesized aspreviously described (Kotake et al. (2007) Nat Chem Biol. 3(9):570-5).100 μL binding reactions were prepared with 50 μL bead slurry and byadding varying concentrations of PB or PB-OH, and after 30 minpre-incubation, 2.5 nM of [³H]-PB was added. The mixture was incubatedfor 30 min, and luminescence signals were read using a MicroBeta2 PlateCounter (PerkinElmer). Prism 7 (Graphpad) was used for non-linearregression curve fitting of the data.

Similar binding profiles were observed for all tested payloads (FIG. 1).In general, specific binding was in the low nanomolar range, suggestingthat all tested payloads are potent SF3b complex binders and promisingcandidate compounds for use in ADCs.

2.2 In Vitro Splicing (IVS)

To evaluate payload activity in a cell-free system, an in vitro splicingassay was performed. The payloads were incubated with nuclear extractsand pre-mRNA substrate minigenes.

HeLa nuclear extract preparation: HeLa S3 cell pellets were resuspendedin hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.2 mMPMSF, 0.5 mM DTT) and the suspension was brought up to a total of 5packed cell volume (PCV). After centrifugation, the supernatant wasdiscarded, and the cells were brought up to 3 PCV with hypotonic bufferand incubated on ice for 10 min. Cells were lysed using a douncehomogenizer and then centrifuged. The supernatant was discarded, and thepellet was resuspended with ½ packed nuclear volume (PNV) of low saltbuffer (20 mM HEPES pH 7.9, 1.5 mM MgCl₂, 20 mM KCl, 0.2 mM EDTA, 25%glycerol, 0.2 mM PMSF, 0.5 mM DTT), followed by ½ PNV of high saltbuffer (same as low salt buffer except 1.4 M KCl). The nuclei weregently mixed for 30 min before centrifuging. The supernatant (nuclearextract) was then dialyzed into storage buffer (20 mM HEPES pH 7.9, 100mM KCl, 0.2 mM EDTA, 20% glycerol, 0.2 mM PMSF, 0.5 mM DTT). Proteinconcentration was determined using NanoDrop 8000 UV-Visspectrophotometer (ThermoFisher Scientific).

IVS: All Ad2-derived sequences (Pellizzoni et al. (1998) Cell95(5):615-24) were cloned into pcDNA3.1(+) vector (Promega) using 5′EcoRI and 3′ XbaI restriction sites. The plasmids were linearized usingXbaI and used as DNA templates in in vitro transcription reactions. TheFtzΔi intron-less plasmid (Luo and Reed (1999) 96(26):14937-42) waslinearized using EcoRI. All RNAs were in vitro transcribed and thenpurified using MEGAScript T7 (Invitrogen) and MegaClear (Invitrogen)kits, respectively. For splicing reactions using Ad2 variant pre-mRNAs,1 μL reactions were prepared using 8 μg nuclear extracts prepared fromHeLa S3, 2 ng pre-mRNA, 0.2 ng FTZΔi, and varying concentrations ofcompounds or DMSO. After a 15 min pre-incubation at 30° C., 1 μLsplicing activation buffer (0.5 mM ATP, 20 mM creatine phosphate, 1.6 mMMgCl₂) was added, and the reactions were incubated for 90 min at 30° C.The reactions were then quenched with 13 μL DMSO, and 25 nL was used forRT-qPCR. RT-qPCR reactions were prepared using TaqMan RNA-to-C_(T)1-step kit (Life Technologies), RNA from splicing reactions, Ad2(forward: ACTCTCTTCCGCATCGCTGT; reverse: CCGACGGGTTTCCGATCCAA; probe:CTGTTGGGCTCGCGGTTG) and Ftz (forward: TGGCATCAGATTGCAAAGAC; reverse:ACGCCGGGTGATGTATCTAT; probe: CGAAACGCACCCGTCAGACG) mRNA primer-probesets. Prism 7 (Graphpad) was used for non-linear regression curvefitting of the formed spliced product and normalized to the control(DMSO) sample. Given that all tested payloads specifically bind to theSF3b complex and demonstrate similar binding profiles (FIG. 1), it washypothesized that all payloads should also modulate splicing to acomparable degree. All payloads significantly modulated splicing ofAd2.2 pre-mRNA (FIG. 2). In the presence of payload, a decrease in theamount of spliced product was observed.

2.3 Cell Viability

HCC1954 (American Type Culture Collection (ATCC)) breast ductalcarcinoma cells were plated at 2000 cells/well in flat bottom 96-welltissue culture plates (Corning) in a total volume of 90 μL tissueculture medium supplemented with 10% fetal bovine serum (ThermoFisherScientific). Cells were treated with a 3-fold serial dilution ofcompound from 200 nM to 0.03 nM. Each concentration was tested intriplicate. At the time of treatment, a plate of untreated cells wasevaluated using CellTiter-Glo®2.0 Luminescent Cell Viability Assayaccording to the manufacturer's recommendations (Promega; #G9241).CellTiter-Glo® 2.0 reagent was added to the medium, incubated, andassayed on an EnVision Multilabel Reader (Perkin Elmer). Valuesrepresent time zero (T0). The number of viable cells following 144 hours(T144) of compound treatment was also determined using theCellTiter-Glo®2.0 Luminescent Cell Viability Assay. Using theluminescence value at time zero (T0), DMSO control growth (C), and testgrowth in the presence of compound (T144), the percentage growth wascalculated at each of the compound concentrations levels. Percentagegrowth inhibition was calculated as: [(T144−T0)/(C−T0)]×100 forconcentrations for which T144>/=T0 or [(T144−T0)/T0]×100 forconcentrations for which T144<T0. The dose response curve plots weregenerated using Prism 7 (Graphpad) and fit using nonlinear regressionanalysis and the log(inhibitor) versus response-variable slope (fourparameters).

Cell viability dose response was determined for all payloads inHER2-amplified HCC1954 breast cancer cells. Most of the tested payloadsexhibited GI₅₀ values (i.e., concentration of compound to cause 50%reduction in cell proliferation) in the single digit nanomolar range,with the exception of poorly permeable payloads such as D25 (FIG. 3).Exemplary permeability data is shown in Table 14.

Example 3

Exemplary payloads evaluated in Example 2 were conjugated to anexemplary anti-HER2 antibody (trastuzumab) via cysteine residues on theantibody. The preparation and evaluation of exemplary anti-HER2 ADCs isdescribed below.

3.1 Antibody

Trastuzumab antibody (“AB185”) (Molina et al. (2001) Cancer Res.61(12):4744-9) was used for the preparation of anti-HER2 ADCs (alsoreferred to herein as SMLAs).

3.2 Bioconjugation

Antibody (trastuzumab) at 10 mg/mL in PBS buffer (pH 7.0) was mixed with5 mM TCEP (2-4 molar equivalents) (ThermoFisher Scientific; #77720) tobreak interchain disulfide bonds. The reaction was gently mixed at 22°C. for 3 hours. Propylene glycol (15% v/v) was then added followed by 8molar equivalents of linker-payload (6 mM stock in DMSO), and thesolution was mixed thoroughly. The reaction was placed onto a rotaryplate in an incubator at 22° C. After a 2-hour conjugation, the reactionmixture was purified to remove unconjugated payload by AKTA GE M150(HiTrap™ 26/10 desalting column; flow rate: 3 mL/min) (GE HealthcareBio-Sciences) into DPBS (pH 7.5). The resulting conjugate wasconcentrated via Amicon ultrafiltration (30 kDa, Ultra-4) (EMDMillipore) and submitted to sterile filtration through a 0.22 μm PVDFdisposable filter (EMD Millipore). The final clear solution was measuredby UV-VIS to determine antibody concentration ([mAb]; mole/L) andconjugated payload concentration ([LD]; mole/L) according to theBeer-Lambert law (A=E*c*l) and the following equations:

A _(280 nm) =E ^(mAb) _(280 nm)*[mAb]*l+E ^(LD) _(280 nm)*[LD]*l

A _(252 nm) =E ^(mAb) _(252 nm)*[mAb]*l+E ^(LD) _(252 nm*[) LD]*l

E ^(mAb) _(280 nm): trastuzumab=213,380 cm⁻¹M⁻¹

E ^(mAb) _(252 nm): trastuzumab=79,112 cm⁻¹M⁻¹

E ^(LD) _(280 nm)=800 cm⁻¹M⁻¹

E ^(LD) _(252 nm)=31,000 cm⁻¹M⁻¹

Abbreviations: c—molar concentration; l—light path length (Nanodrop: 0.1cm); E—molar extinction coefficient; A—absorbance.

3.3 Biophysical Characterization

The drug-to-antibody ratio (DAR), percent aggregation, and percentunconjugated payload was analyzed for exemplary anti-HER2 ADCs by liquidchromatography-mass spectrometry (LC/MS), size exclusion chromatography(SEC), and reverse-phase high-performance liquid chromatography (HPLC),respectively. In general, conjugates contained less than 2% free drugand contained less than 10% aggregate.

3.3.1 LC/MS Analysis—DAR

LC/MS analysis was performed using an Agilent 1290 UPLC systeminterfaced to an Agilent G6224A Accurate Mass TOF mass spectrometer.Conjugate was deglycosylated with PNGase F (New England Biolabs;#P0705L) for 4 hours at 37° C., denatured with 8 M Gdn-HCl (Sigma;#G9284), and finally separated into light and heavy chain domains usingDTT (5 mM final concentration) (Promega; #V3151). The prepared samplewas injected onto an Agilent PLRP-S column (2.1×150 mm, 8 μm) and elutedwith a gradient of 25% B to 50% B over 28 min at room temperature (RT).Mobile phase A was water with 0.05% TFA, mobile phase B was acetonitrilewith 0.04% TFA, and the flow rate was 1 mL/min. DAR was calculated fromthe deconvoluted mass spectrum by weighted averaging the intensities ofthe unconjugated and drug conjugated peaks for the light chain (L0 orL1) and heavy chain (H0, H1, H2, and H3). The total DAR of the intactconjugate was calculated using the equation:(DAR_(LC)*2)+(DAR_(HC)*2)=total DAR. DAR values for exemplary anti-HER2ADCs are reported in Tables 10-14.

3.3.2 SEC Analysis—Aggregation

Size exclusion chromatography was performed using a TOSON-G3000SWXL(#008541) column in 0.2 M potassium phosphate (pH 7) with 0.25 mMpotassium chloride and 15% (v/v) IPA at a flow rate of 0.75 mL/min. Thepeak area absorbance at 280 nm was determined for the high molecularweight and monomeric conjugate components by area under the curveintegration. Percent monomer for exemplary anti-HER2 ADCs is reported inTable 10.

3.3.3 HPLC Analysis—Free Drug

Conjugate was precipitated with 10 volumes of acetonitrile on ice for 2hours and spun down. Supernatants containing residual unconjugatedpayload were then injected onto an Agilent Poroshell 120 SB-C18 120Acolumn (4.6×100 mm, 2.7 μm) and eluted with a gradient of 45% B to 70% Bover 10 min at RT. Mobile phase A was 100% water, mobile phase B was100% acetonitrile, and the flow rate was 0.6 mL/min with detection at252 nm. The amount of residual free drug was quantified via UV detectionwith comparison to the external standard curve of unconjugatedlinker-payload. Percent free drug for exemplary anti-HER2 ADCs isreported in Table 10.

3.4 Binding Characterization 3.4.1 FACS Binding to Target-Positive Cells

Binding of unconjugated anti-HER2 antibody and anti-HER2 ADCs totarget-positive cells was evaluated by flow-cytometry using indirectimmunofluorescence. JIMT1 cells (DSMZ), a breast cancer cell line thatendogenously expresses HER2, were plated (5×10⁴ cells/well) in av-bottom 96-well plate (Greiner Bio-One) and incubated for 2 hours at 4°C. with the test compounds diluted to various concentrations in assaymedium (RPMI-1640 supplemented with 10% (w/v) fetal bovine serum albumin(Thermo Fisher Scientific)). The cells were then washed with PBS+2% FBS(FACS buffer), and stained with phycoerythrin-labeled (PE) goatanti-human immunoglobulin G (IgG) antibody (Invitrogen) for 40 min at 4°C. in the dark. Cells were washed with cold FACS buffer and fixed withFluroFix buffer (Biolegend) for 30 min at room temperature. Fixative waswashed off with FACS buffer. Fixed cells were analyzed for the geometricmean fluorescence of PE using an LSRFortessa flow cytometer (BDBioscience). Trastuzumab and T-DM1 (DM1 conjugated to trastuzumab) wereincluded as controls.

All anti-HER2 ADCs demonstrated robust binding to HER2 in JIMT1 cells.ADC binding was comparable to binding of trastuzumab and T-DM1 (FIG. 4).This suggests that conjugation to payload does not affect antigenbinding affinity of the antibody.

3.5 In Vitro Analysis 3.5.1 Cell Viability

Anti-HER2 ADCs were tested in several HER2-amplified cell lines fortheir ability to inhibit cell growth. HCC1954 (ATCC), 2000 cells/well),N87 (ATCC, 4000 cells/well), SKBR3 (ATCC, 3000 cells/well), and MCF7(ATCC, 1500 cells/well) cell lines were used. Cell viability analysiswas performed as described in section 2.3.

Surprisingly, not all ADCs were active in HCC1954 cells, despite havingsimilar binding profiles (FIG. 1) and payloads with similar biochemicalproperties. ADCs with certain linkers (e.g., ADL10) and/or certainpayloads (e.g., D14) were less capable of inhibiting cell growth,whereas ADCs with alternate linkers (ADL1, ADL5, ADL12) and/or payloads(e.g., D1, D25, D2, D4) were more potent in HCC1954 cells (FIG. 5A andTables 10, 11, and 14). These trends were generally observed across celllines (HCC1954, N87, and SKBR3) and indications (HCC1954 and SKBR3 arehigh HER2 breast cancer lines; N87 is a high HER2 gastric cancer line)(FIG. 5B-C and Tables 11 and 12). Moreover, in general, traditionalnon-cleavable linkers such as ADL10 did not deliver splicing modulatorpayloads efficiently. However, small modifications in length to the samelinkers rendered ADCs much more potent, e.g., compare cell viabilitydose response of AB185-ADL10-D1 to AB185-ADL12-D1 (FIG. 5A-C).

To ensure activity of anti-HER2 ADCs is antigen-dependent, HER2-negativeMCF7 cells were treated. None of the tested ADCs were active at the sameconcentrations that robustly targeted HER2-positive cells (FIG. 6 andTable 13). This suggests that anti-HER2 ADC activity isantigen-dependent.

TABLE 10 Characterization of exemplary anti-HER2 ADCs CTGIo CTGIo CTGIoFree GMean GMean Mean SMLA Batch Payload Percent Drug Concentration GI50(nM) LD50 (nM) MinResponse % ID Class Linker Linker Name DAR Monomer (%)(mg/ml) HCC1954.1 HCC1954.1 HCC1954.1 AB185- Plad D ADL0010- maleimido2.000 99.000 <1 0.730 >200.000 >200.000 58.601 ADL10-D8 01 caproylAB185- Plad D ADL0010- maleimido 3.600 97.000 <1 3.600 25.502 >200.00023.963 ADL10-D1 01 caproyl AB185- Aryl Plad ADL0005- mc-Val-Ala- 3.96098.000 <1 1.400 2.668 >160.000 14.873 ADL5-D17 01 PAB (Qamine) AB185-Aryl Plad ADL0001- mc-Val-Cit- 4.400 95.000 <1 0.550 2.086 158.245ADL1-D16 01 PABC AB185- Aryl Plad ADL0005- mc-Val-Ala- 4.400 98.000 <11.200 0.943 >200.000 ADL5-D26 01 PAB (Qamine) A8185- Aryl Plad ADL0001-mc-Val-Cit- 4.340 88.000 <1 4.200 0.925 >200.000 1.271 ADL1-D14 01 PABCAB185- Aryl Plad ADL0005- mc-Val-Ala- 4.000 98.000 <1 0.7800.912 >200.000 −45.388 ADL5-D15 01 PAB (Qamine) AB185- Plad D ADL0005-mc-Val-Ala- 3.700 96.000 <1 0.880 0.806 >93.000 −30.929 ADL5-D11 01 PAB(Qamine) AB185- Aryl Plad ADL0001- mc-Val-Cit- 8.000 95.000 <1 0.5700.466 >200.000 −25.859 ADL1-D33 01 PABC AB185- Plad D ADL0001-mc-Val-Cit- 3.000 99.000 <1 0.840 0.443 >200.000 −7.520 ADL1-D22 01 PABCAB185- Plad D ADL0012- mc-(CH2)6- 3.600 98.600 <1 1.060 0.432 >200.00018.569 ADL12-D2 01 (non-cleavable) AB185- Plad B ADL0001- mc-Val-Cit-5.000 98.000 <1 1.200 0.289 18.948 −94.860 ADL1-D13 01 PABC AB185- PladB ADL0005- mc-Val-Ala- 6.600 97.000 <1 3.230 0.278 >200.000 −47.381ADL5-D19 01 PAB (Qamine) AB185- Plad D ADL0005- mc-Val-Ala- 4.020 97.000<1 0.650 0.254 >200.000 −2.273 ADL5-D10 01 PAB (Qamine) AB185- Plad DADL0006- mc-Val-Ala- 5.200 98.000 <1 0.500 0.244 4.206 −94.511 ADL6-D901 PABC AB185- Plad D ADL0005- mc-Val-Ala- 4.140 99 <1 4.50.206 >200.000 −12.239 ADL5-D2 01 PAB (Qamine) AB185- Plad D ADL0001-mc-Val-Cit- 7.200 97.000 <1 3.410 0.188 >200.000 −55.079 ADL1-D8 01 PABCAB185- Plad D ADL0005- mc-Val-Ala- 4.300 99.000 <1 5.440 0.179 >200.000−18.586 ADL5-D2 01 PAB (Qamine) AB185- Plad D ADL0015- mal-CH2CH2— 3.30098.800 <1 1.600 0.170 >200.000 −23.513 ADL15-D2 01 O—CH2CH2—(non-cleavable) AB185- Plad D ADL0012- mal-(CH2)6- 3.200 98.600 <1 1.3900.142 >200.000 −57.399 ADL12-D20 01 (non-cleavable) AB185- Plad DADL0012- mal-(CH2)6- 3.300 98.800 <1 1.620 0.126 >200.000 −34.649ADL12-D1 01 (non-cleavable) T-DM1 DM1 ADL0019- SMCC 3.300 98.000 <13.300 0.118 2.759 −47.952 01 AB185- Plad D ADL0005- mc-Val-Ala- 8.500 98<1 5.1 0.086 2.918 −63.909 ADL5-D2 01 PAB (Qamine) AB185- Plad DADL0006- mc-Val-Ala- 8.000 97.000 <1 1.200 0.081 2.877 −85.692 ADL6-D101 PABC AB185- Plad D ADL0012- mal-(CH2)6- 3.000 98.000 <1 2.860 0.0812.148 −68.999 ADL12-D1 01 (non-cleavable) AB185- Plad D ADL0012-mal-(CH2)6- 4.300 97.000 <1 0.120 0.078 >80.000 28.071 ADL12-D22 01(non-cleavable) AB185- Plad D ADL0001- mc-Val-Cit- 8.000 98.000 <1 3.6000.073 1.949 −67.199 ADL1-D4 01 PABC AB185- Plad D ADL0015- mal-CH2CH2—3.200 98.800 <1 0.950 0.062 >200.000 −57.829 ADL15-D1 01 O—CH2CH2—(non-cleavable) AB185- Plad D ADL0014- mal-CH2CH2— 3.500 98.600 <1 0.7400.055 >200.000 −56.889 ADL14-D1 01 (non-cleavable) AB185- Plad DADL0001- mc-Val-Cit- 2.800 98.000 <1 1.200 0.050 >200.000 −57.212ADL1-D4 01 PABC AB185- Plad D ADL0001- mc-Val-Cit- 3.900 99 <1 5.2 0.0460.374 −78.519 ADL1-D1 01 PABC AB185- Plad D ADL0001- mc-Val-Cit- 4.00098.000 <1 3.500 0.042 0.447 −77.970 ADL1-D1 01 PABC AB185- Plad DADL0005- mc-Val-Ala- 6.000 98.000 <1 0.170 0.038 >113.000 −46.265ADL5-D25 Zwitt 01 PAB (Qamine) AB185- Plad D ADL0001- mc-Val-Cit- 7.70098.000 <1 4.200 0.034 0.120 −87.001 ADL1-D1 01 PABC

TABLE 11 Exemplary anti-HER2 ADCs - HCC1954 cells ADC Payload GI50 LD50Rmin GI50 LD50 Rmin Sample DAR (nM) (nM) (%) (nM) (nM) (%) AB185- 3.10.064 4.86 −78.393 1.853 5.485 −75.579 ADL12-D1 AB185- 7.2 0.075 1.01−72.765 2.638 11.175 −80.463 ADL1-D8 T-DM1 3.3 0.083 >200 −64.227 AB185-4.04 0.089 2.078 −74.165 1.853 5.485 −75.579 ADL1-D1 AB185- 4.9 0.1451.217 −75.638

6.374 −81.34 ADL1-D4 AB185- 4.1 0.18 >200 −36.513 0.6 2.758 −78.732ADL5-D2 AB185- 4 0.503 >200 −20.387 0.358 2.037 −84.412 ADL5-D15 AB185-3.8 5.994 >200 −8.011 0.208 0.787 −90.055 ADL1-D18 AB185- 3.6 6.129 >20018.763 0.6 2.758 −78.732 ADL12-D2 AB185- 3.5 6.642 >200 8.138 1.8535.485 −75.579 ADL10-D1 AB185- 6.3 0.038 >113.000 −51.503 167.54 >20026.882 ADL5-D25 AB185 N/A >200 >200 76.916

indicates data missing or illegible when filed

TABLE 12 Exemplary anti-HER2 ADCs - N87 cells SMLA Payload GI50 LD50Rmin GI50 LD50 Rmin Sample DAR (nM) (nM) (%) (nM) (nM) (%) AB185- 4.90.093 >200 −40.056 3.241 38.774 −81.775 ADL1-D4 AB185- 4 0.106 >200−50.886 0.41 >200 −68.045 ADL5-D15 AB185- 6.3 0.11 133 −43.398 >200 >20040.364 ADL5-D25 AB185- 3.1 0.111 >200 −41.228 7.041 >200 −40.041ADL12-D1 AB185- 3.8 0.113 >200 −39.509 0.275 2.993 −92.127 ADL1-D18AB185- 4.04 0.163 >200 −41.075 7.041 >200 −40.041 ADL1-D1 T-DM1 3.30.168 >200 −20.785 AB185- 7.2 0.172 >200 −31.708 8.844 >200 −34.052ADL1-D8 AB185- 4.1 0.206 >200 −51.328 0.749 >200 −72.706 ADL5-D2 AB185-3.6 0.243 >200 −9.79 0.749 >200 −72.706 ADL12-D2 AB185- 4.3 0.296 >200−26.143 2.925 >200 −52.662 ADL1-D14 AB185- 3.5 1.531 >200 30.7597.041 >200 −40.041 ADL10-D1 AB185 N/A >200 >200 77.029

TABLE 13 Exemplary anti-HER2 ADCs - MCF7 cells SMLA Payload GI50 LD50Rmin GI50 LD50 Rmin Sample DAR (nM) (nM) (%) (nM) (nM) (%) T-DM1 3.338.503 >200 3.876 AB185- 3.1 57.818 >200 −30.172 16.998 135.42 −63.414ADL12-D1 AB185- 6.3 80.856 113 28.12 >200 >200 65.639 ADL5-D25 AB185-4.9 189.392 >200 43.848 11.416 96.46 −60.923 ADL1-D4 AB185-4.1 >200 >200 56.182 2.851 30.318 −66.915 ADL5-D2 AB185- 7.2 >200 >20061.169 29.614 181.521 −62.957 ADL1-D8 AB185- 3.8 >200 >200 48.851 0.8837.697 −68.226 ADL1-D18 AB185- 4 >200 >200 49.619 0.961 130.398 −68.503ADL5-D15 AB185- 3.6 >200 >200 48.868 2.851 30.318 −66.915 ADL12-D2AB185- 3.5 >200 >200 52.699 16.998 135.42 −63.414 ADL10-D1 AB185-4.3 >200 >200 49.325 5.288 77.198 −61.886 ADL1-D14 AB185- 4.04 >200 >20059.181 16.998 135.42 −63.414 ADL1-D1 AB185 N/A >200 >200 72.747

3.5.2 Caco-2 Permeability of ADC Payload

Caco-2 cells were cultured for 21 days in transwell 24-well plates at37° C., 95% humidity, 5% CO₂. Integrity of cell monolayer was confirmedby TEER (transepithelial electrical resistance) and Lucifer yellow.Payloads were spiked in duplicate at 10 μM, separately, on both sides ofthe cell monolayer. Permeability rates from the apical to basolateral(A-B) direction and the basolateral to apical (B-A) direction weredetermined by sampling aliquots from both chambers immediately aftertreatment (t=0) and following incubation for 2 hours. Samples wereprotein precipitated with organic solvent containing internal standardand analyzed by LC-MS/MS (SCIEX; API 5500). The area ratio responses ofpayload/internal standard over time in both directions were used togenerate permeability (cm/sec) values. Efflux ratio was calculated bydividing B−A/A−B. Control compounds for low and high permeability andefflux behaved as expected. Permeability values are reported in Table14.

3.5.3 Chemical Stability of ADC Payload

Payloads were incubated in Mcilvane (Citrate-Phosphate) buffer, pH 5.5(Boston Bioproducts; #BB-2466) at a final concentration of 20 μM (lessthan 0.5% DMSO from stock solution). The payload solution and theinternal standard were pipetted into 96-well plates, ran on UPLC (WatersAquity H class), and analyzed for initial chromatographic signal (t=0).The column was a Waters UPLC HSS T3 1.8 μm 2.1×50 mm column(#186003538). A gradient of mobile phase A from 95% to 10% was employedover 1 min, where A was 0.1% formic acid in water and mobile phase B was0.1% formic acid in acetonitrile (flow rate 0.9 mL/min). The remainderof the payload solution was kept in a plate shaker at 37° C. (EppendorfThermoMixer). Sample analyses by UPLC were repeated at 24, 72, and 96hours post-incubation at 37° C. The area ratio response of the payloadand internal standard was determined for three time points: time 0, day1, and either day 3 or day 4. Time 0 was set to 100. The area ratioresponses of the later time points were compared to time 0. Percentremaining was calculated as follows: (Area Ratio day X/Area Ratio time0)*100=% remaining. The slope of the line was calculated in Excelcomparing the log of % remaining and time point. Half-lives werecalculated in Excel by ln(2)/slope and are reported in Table 14.

TABLE 14 Characterization of exemplary anti-HER2 ADCs and correspondingpayloads Payload SF3B1 binding and mRNA splice modulation SPA-ATS qPCRqPCR SMLA activity Gmean IVS-ATS IVS-ATS CTGIo CTGIo IC50 (nM) GmeanGmean Gmean Gmean Rmin (%) SF3B1 IC50 (nM) IC50 (nM) SMLA Batch GI50(nM) LD50 (nM) (max Payload (WT) Ad2.1 AD2.2 ID Linker DAR HCC1954.1HCC1954.1 lethality) Class Mass HELA.2 HELA.2 HELA.2 AB185- ACL0005-

0.038 >113.000

Plad D 722.917

243.748

ADL5-D25 01 Zwitt AB185- ACL0001- 4 0.042 0.447

Plad D

15.5853 ADL1-D1 01 AB185- ACL0001- 2.8 0.05 >200.000

Plad D

8.778 20.887 17.321 ADL1-D4 01 AB185- ACL0014- 3.5 0.055 >200.000

Plad D 636.827

15.853 ADL14-D1 01 AB185- ACL0015- 3.2 0.062 >200.000

Plad D

15.853 ADL15-D1 01 AB185- ACL0001- 7.2 0.075 1.01

Plad D 664.837 5.821 11.403 8.034 ADL1-D8 01 AB185- ACL0012- 4.30.078 >80.000 27.157 Plad D 708.89

49.131 ADL12-D22 01 Zwitt AB185- ACL0012- 3.1 0.081 2.148

Plad D

15.853 ADL12-D1 01 AB185- ACL0005- 3.8 0.081 2.877

Plad D 636.827

15.853 ADL6-D1 01 T-DM1 SMCC 3.3 0.118 2.759

DM1

>1200.000

AB185- ACL0012- 3.3 0.126 >200.000

Plad D

ADL12-D21 01 AB185- ADL0012- 3.2 0.142 >200.000

Plad D

18.251 10.829 8.748 ADL12-D20 01 AB185- ADL0015- 3.3 0.17 >200.000

Plad D

15.533 19.133 15.342 ADL15-D2 01 AB185- ADL0005- 4.1 0.179 >200.000

Plad D

19.133 15.342 ADL5-D2 01 AB185- ADL0006-

0.244

Plad B

2.117 20.224

ADL6-D9 01 AB185- ADL0005- 4 0.254 >200.000 −7.125 Plad D

9.228

ADL5-D10 01 AB185- ADL0001-

18.948

Plad B

2.065 11.287

ADL1-D13 01 AB185- ADL0012-

0.432 >200.000 17.824 Plad D

19.133

ADL12-D2 01 AB185- ADL0001- 2.9 0.443 >200.000 27.157 Plad D

ADL1-D22 01 Zwitt AB185- ADL0001- 8

>200.000

630.758

72.404 ADL1-D33 01 Zwitt AB185- ADL0005- 3.7

>93.000 −57.844 Plad D

10.545

ADL5-D11 01 AB185- ADL0005- 4

>200.000 −50.337

ADL5-D15 01 AB185- ADL0001- 4.3

>200.000

1.924 10.022

ADL1-D14 01 AB185- ADL0005- 4.3

>200.000

18.838 14.314 ADL5-D26 01 AB185- ADL0001- 4.3

ADL1-D16 01 AB185- ADL0005- 4

>100.000 12.42

587.733 1.82 17.943

ADL5-D17 01 AB185- ADL0001- 3.8

>200 −8.011 Plad B

12.709 ADL1-D18 01 AB185- ADL0010- 3.5

>200

Plad D

ADL10-D1 01 Payload cell permeability Payload Payload cell potency andPermeability Permeability chemical lethality Mean Mean stabilityCTGIo-ATS CTGIo-ATS CTGIo-ATS Caco-2 Caco-2 Stability Gmean Gmean MeanA-B B-A Mean GI50 (nM) LD50 (nM) MinResponse % Perm Perm Stability SMLABatch 72 h 72 h 72 h (10e−6 (10e−6 Efflux t½ ID THP1.1 THP1.1 THP1.1cm/s) cm/s) Ratio pH 5.5 (d) AB185- 348.114 2.078.811

0.137 0.349

5 ADL5-D25 AB185- 11.085 41.408 −100.615 0.14 2.05

ADL1-D1 AB185- 10.943 33.329

0.1 1.79 17.9 4.4 ADL1-D4 AB185-

41.408

0.14 2.05

3.85 ADL14-D1 AB185-

41.408

0.14 2.05

3.95 ADL15-D1 AB185-

0.128 2.027 15.875 3.525 ADL1-D8 AB185- 223.348

0.14

1.143 ADL12-D22 AB185-

41.408

0.14 2.05

ADL12-D1 AB185- 11.085 41.408 −100.815 0.14 2.05 14.843 3.85 ADL6-D1T-DM1

3.287

28.0

1.3 AB185- ADL12-D21 AB185- 38.338

0.38

>7000 ADL12-D20 AB185- 2.015 11.708 −100.218

14.2

4 ADL15-D2 AB185- 2.015 11.708 −100.218

14.2

4 ADL5-D2 AB185- 0.87 3.56

1.15

14.008 <1.000 ADL6-D9 AB185- 1.819 8.454

1.81

12.983 >7.000 ADL5-D10 AB185- 0.892 3.358 −99.941

17.17 19.078 <1.000 ADL1-D13 AB185- 2.015 11.708 −100.218

14.2 22.758 4 ADL12-D2 AB185- 223.348

−78.118 0.14

1.143 ADL1-D22 AB185- 196.752

−53.128 0.13 0.53 4.077 ADL1-D33 AB185-

−100.481 0.87

3.75 ADL5-D11 AB185- 1.017

7.843 >7.000 ADL5-D15 AB185-

>7.000 ADL1-D14 AB185-

2.578 >7.000 ADL5-D26 AB185-

0.72

>7.000 ADL1-D16 AB185- 2.827 24.908

20.25 18.18

ADL5-D17 AB185-

1.19 27.4

ADL1-D18 AB185-

0.14 2.05

ADL10-D1

indicates data missing or illegible when filed3.5.4 in-Cell Splicing PD Assay

To interrogate the mechanism of action of exemplary anti-HER2 ADCs,splicing of the SLC25A19 mature transcript was examined in HCC1954 cellstreated with increasing concentrations of ADC.

HCC1954 cells (ATCC) were plated in phenol-red free RPMI+10% FBS media(ATCC) at 1×10⁵ cells per well at 90 μL per well. Cells were treatedwith conjugates in a 3-fold dilution dose-response. After 24 hours or 6hours, respectively, cells were lysed with 50 μL of CL buffer (IgePalCA-630, 5M NaCl, 1M Tris HCl 1M pH 7.4 in water) containing 25 μL/mL ofRNAsin (Promega) and incubated for 45 min at RT on a rocker. Resultingmixture (0.5 μL) was used to assess splicing modulation in a Taqman FastVirus 1-Step MasterMix (Applied Biosystems) reverse transcription PCRreaction with the following Taqman primers according to themanufacturer's recommendations: SLC25A19 (Invitrogen, Hs00222265_m1);RPLPO (Invitrogen, Hs99999902_m1); 18S (Invitrogen, Hs99999901_s1).

T-DM1 control (trastuzumab with a microtubule disrupting agent aspayload) did not affect SLC25A19 splicing (FIG. 7). In contrast,anti-HER2 ADCs that demonstrated potent growth inhibition activity indifferent tested cell lines (FIG. 5) also demonstrated potent splicingmodulation, indicating an on-target mechanism of action (FIG. 7).Likewise, anti-HER2 ADCs that exhibited lower potency in cell viabilityassays were less potent in terms of splicing modulation.

3.5.5 Bystander Cell Killing Assay

The ability of exemplary anti-HER2 conjugates to kill bothtarget-positive cells and neighboring target-negative cells was measuredin a bystander cell killing assay. NCI-H1568, non-small cell lung cancercells (ATCC) were engineered to express luciferase. cDNA was synthesizedat GeneArt (ThermoFisher Scientific) and cloned into a pLVX-EF1adestination vector (ThermoFisher Scientific). Separately, NCI-H1568 wereengineered to express human HER2 (cloned into pLenti6.3/V5-DEST,ThermoFisher Scientific) utilizing the TransiT® viral transfectionsystem according to manufacturer's instructions (Mirus Bio LLC;#MIR6003). To assess the bystander activity of conjugates, 2000target-positive (HER2-transduced) NCI-H1568 cells and 2000target-negative (luc-tagged) NCI-H1568 cells were plated together inclear 96-well round bottom plates (Corning) and incubated withconjugates dosed at a 3-fold serial dilution in triplicate, 30-0.005 nM.At the time of treatment, a plate of untreated cells was assayed usingOneGlo® Luciferase Assay System (Promega) and CellTiter-Glo®2.0Luminescent Cell Viability Assay System (Promega) according to themanufacturer's recommendations, to establish a time zero (T0). After 6days in culture, the remaining cell population was analyzed by OneGlo®Luciferase Assay System (Promega) to measure the fraction oftarget-negative cells surviving. Overall viability was measured byCellTiter-Glo®2.0 Luminescent Cell Viability Assay System (T144). Doseresponse curves were calculated as described in section 2.3.

The design of the bystander killing assay allows tracking of onlytarget-negative cells, the elimination of which relies on uptake of thecatabolite released by neighboring target-positive cells. As discussedabove, cells are plated under three conditions: (1) target-negative(luc-tagged) cells alone; (2) target-positive cells alone; and (3)co-culture of target-negative and target-positive cells. When the platesare read with CellTiter-Glo® reagent, viability of all cell populationscan be tracked to confirm antigen-dependent ADC activity. In contrast,when OneGlo® reagent is used, only the target-negative (luc-tagged)cells will have a signal.

Target-negative (luc-tagged) cells were not sensitive to treatment withanti-HER2 ADCs when cultured on their own (FIG. 8). However, whentarget-negative cells were cultured with target-positive cells,treatment with anti-HER2 ADCs resulted in increased killing oftarget-negative cells. These data suggest that target-negative cells arekilled more effectively by anti-HER2 ADCs when co-cultured withtarget-positive cells, referred to herein as bystander killing.Bystander killing is distinguishable from off-target killing, which isdefined as the killing of target (antigen)-negative cells on their own,in the absence of and independent of co-culturing with target-positivecells.

In this experiment, AB185-ADL5-D2 demonstrated an increased ability tokill an antigen heterogenous cancer cell population as compared toAB185-ADL1-D1. Without wishing to be bound by theory, this difference inpotency may be due to the addition of one methyl group in D2, whichgives a three-fold increase in payload cell potency and permeability ascompared to D1 (see Table 14). The D2 ADC treated an antigenheterogenous cancer cell population as effectively as theAB185-MC-Val-Cit-MMAE control ADC.

3.6 In Vivo Analysis

Exemplary anti-HER2 ADCs that exhibited potent splicing modulation andcell growth inhibition were assessed in vivo.

3.6.1 HCC1954 Xenograft Efficacy Study

To investigate the efficacy of exemplary anti-HER2 ADCs in a mousexenograft model, HCC1954 (ATCC) breast ductal carcinoma cells (10×10⁶cells/100 μL/1:1 RPMI:Matrigel volume) were subcutaneously implantedinto the flank of female CB17-SCID mice. Mice were treated with singlebolus IV doses of conjugates (formulated in DPBS, pH 7.4) or vehiclecontrol. The animals were dosed at the amounts indicated in FIG. 9. Allanimals were monitored for tumor growth and body weight twice weeklyuntil they reached either of the following endpoints: (1) excessivetumor volume (calculated by using the ellipsoid formula:(length×width²)/2); or (2) development of any health problems such asexcessive body weight loss. All animal studies were carried outaccording to the H3 Biomedicine Guide for the Care and Use of LaboratoryAnimals.

Treatment with anti-HER2 ADC (AB185-ADL5-D2 or AB185-ADL1-D1) at asingle dose as low as 2 mg/kg resulted in significant tumor regression(FIG. 9). A dose response for the conjugated payload was also observed,such that anti-HER2 ADCs with higher DAR values (DAR ˜4) demonstratedincreased efficacy and slower tumor regrowth relative to dose-matchedantibody T-DM1 control. Additionally, payload targeted to the tumor viaan ADC was more potent relative to the corresponding unconjugatedpayload.

Example 4

To determine whether the properties of the exemplary anti-HER2 ADCsdescribed in Example 3 could be extrapolated to other ADCs targetingalternate antigens and/or indications, select payloads were conjugatedto an exemplary anti-CD138 antibody (B-B4) and an exemplary anti-EPH2Aantibody (1C1). CD138 is expressed on malignant plasma cells, e.g., inmultiple myeloma, while EPH2A is more generally expressed in a varietyof malignancies. The preparation and evaluation of exemplary anti-CD138and anti-EPH2A ADCs is described below.

4.1 Antibodies

B-B4 antibody (“AB205”) (Tassone et al. (2004) Blood 104:3688-96) and101 antibody (“AB206”) (Jackson et al. (2008) Cancer Res.68(22):9367-74) were used for the preparation of anti-CD138 ADCs andanti-EPH2A ADCs, respectively.

4.2 Bioconjugation

Antibody (B-B4 or 101) at 10 mg/mL in PBS buffer (pH 7.0) was mixed with5 mM TCEP (2-4 molar equivalents) (ThermoFisher Scientific; #77720) tobreak interchain disulfide bonds. The reaction was gently mixed at 22°C. for 3 hours. Propylene glycol (15% v/v) was then added followed by 8molar equivalents of linker-payload (6 mM stock in DMSO), and thesolution was mixed thoroughly. The reaction was placed onto a rotaryplate in an incubator at 22° C. After a 2-hour conjugation, the reactionmixture was purified to remove unconjugated payload by AKTA GE M150(HiTrap™ 26/10 desalting column; flow rate: 3 mL/min) (GE HealthcareBio-Sciences) into DPBS (pH 7.5). The resulting conjugate wasconcentrated via Amicon ultrafiltration (30 kDa, Ultra-4) (EMDMillipore) and submitted to sterile filtration through a 0.22 μm PVDFdisposable filter (EMD Millipore). The final clear solution was measuredby UV-VIS to determine antibody concentration ([mAb]; mole/L) andconjugated payload concentration ([LD]; mole/L) according to theBeer-Lambert law (A=E*c*I) and the following equations:

A _(280 nm) =E ^(mAb) _(280 nm)*[mAb]*l+E ^(LD) _(280 nm)*[LD]*l

A _(252 nm) =E ^(mAb) _(252 nm)*[mAb]*l+E ^(LD) _(252 nm)*[LD]*l

E ^(mAb) _(280 nm) : B−B4=224,320 cm⁻¹M⁻¹

1C1=215,380 cm⁻¹M⁻¹

E ^(mAb) _(252 nm) : B−B4=83,670 cm⁻¹M⁻¹

1C1=80,337 cm⁻¹M⁻¹;

E ^(LD) _(280 nm)=800 cm⁻¹M⁻¹

E ^(LD) _(252 nm)=31,000 cm⁻¹M⁻¹

Abbreviations: c—molar concentration; l—light path length (Nanodrop: 0.1cm); E—molar extinction coefficient; A—absorbance.

4.3 Biophysical Characterization

DAR, percent aggregation, and percent unconjugated payload was analyzedfor exemplary anti-CD138 and anti-EPHA2 ADCs by liquidchromatography-mass spectrometry (LC/MS), size exclusion chromatography(SEC), and reverse-phase high-performance liquid chromatography (HPLC),respectively. All conjugates described herein contained less than 2%free drug and contained less than 10% aggregate.

4.3.1 LC/MS Analysis—DAR

LC/MS analysis was performed as described in section 3.3.1. DAR valuesfor exemplary anti-CD138 and anti-EPHA2 ADCs are reported in Tables15-18.

4.3.2 SEC Analysis—Aggregation

SEC analysis was performed as described in section 3.3.2. Percentmonomer for exemplary anti-CD138 and anti-EPHA2 ADCs is reported inTables 15 and 17.

4.3.3 HPLC Analysis—Free Drug

HPLC analysis was performed as described in section 3.3.3. Percent freedrug for exemplary anti-CD138 and anti-EPHA2 ADCs is reported in Tables15 and 17.

4.4 In Vitro Analysis 4.4.1 Cell Viability

Anti-CD138 ADCs were tested in the CD138-expressing MOLP8 multiplemyeloma cell line. Similarly, anti-EPHA2 ADCs were tested in theEPHA2-expressing PC3 prostate cancer cell line. Cell viability analysiswas performed as described in section 2.3.

Payload conjugation to anti-CD138 antibody (FIG. 10 and Table 16) andanti-EPHA2 antibody (FIG. 11 and Table 18) resulted in similar trends asthose discussed in section 3.5.1 for anti-HER2 ADCs. In particular, ADCswith certain linkers (e.g., ADL12) and/or payloads (e.g., D1, D25, D4)demonstrated the highest potency in antigen-expressing cells.Consistently, alternate payloads (e.g., D14) were less capable ofinhibiting cell growth via antibody-mediated delivery, despite beingpotent small molecules.

TABLE 15 Characterization of exemplary anti-CD138 ADCs Free SMLA BatchPayload Percent Drug Concentration ID Class Linker DAR Monomer (%)(mg/ml) AB205- Aryl Plad ADL0001 2.900 92.000 <2 1.010 ADL1-D14 AB205-Aryl Plad ADL0005 2.800 95.000 <2 1.060 ADL5-D15 AB205- Plad B ADL00054.900 95.000 <2 0.880 ADL5-D19 AB205- Plad D ADL0001 5.000 98.000 <20.620 ADL1-D1 AB205- Plad D ADL0001 4.800 98.000 <2 1.010 ADL1-D4 AB205-Plad D ADL0005 6.400 98.000 <2 0.890 ADL5-D25 Zwitt AB205- Plad DADL0010 3.400 96.000 <2 0.750 ADL10-D1 AB205- Plad D ADL0012 3.80097.000 <2 0.830 ADL12-D1 AB205- Plad D ADL0012 3.300 97.000 <2 0.970ADL12-D2

TABLE 16 Exemplary anti-CD138 ADCs - MOLP8 cells SMLA Payload GI50 LD50Rmin GI50 LD50 Rmin Sample DAR (nM) (nM) (%) (nM) (nM) (%) AB205- 5<0.061 0.656 −100.099 6.885 25.712 −100.248 ADL1-D1 AB205- 2.80.268 >400 2.419 0.776 9.178 −100.198 ADL5-D15 AB205- 4.9 <0.061 >400−46.23 0.84 4.58 −62.2 ADL5-D19 AB205- 3.4 0.08 >400 −25.099 6.88525.712 −100.248 ADL10-D1 AB205- 3.8 <0.061 0.168 −99.851 6.885 25.712−100.248 ADL12-D1 AB205- 3.3 <0.061 >400 −53.125 0.316 5.035 −100.198ADL12-D2 AB205- 3 <0.061 >400 −74.008 1.9 8.5 −61 ADL1-D14 AB205- 4.8<0.061 0.535 −99.306 1.151 12.99 −101.935 ADL1-D4 AB205- 6.4 <0.06111.669 −76.736 149.874 >400 5.94 ADL5-D25

TABLE 17 Characterization of exemplary anti-EPHA2 ADCs Free SMLA BatchPayload Percent Drug Concentration ID Class Linker DAR Monomer (%)(mg/ml) AB206- Plad D ADL0001 5.600 99.000 <2 2.060 ADL1-D1 AB206- PladD ADL0005 6.300 99.000 <2 1.900 ADL5-D2 AB206- Plad D ADL0001 6.00095.000 <2 1.140 ADL1-D4 AB206- Plad D ADL0001 6.500 95.000 <2 1.660ADL1-D8 AB206- Aryl Plad ADL0001 3.600 80.000 <2 1.620 ADL1-D14 AB206-Plad B ADL0005 4.600 97.000 <2 0.960 ADL5-D19 AB206- Aryl Plad ADL00054.200 95.000 <2 0.820 ADL5-D15 AB206- Plad D ADL0005 5.000 96.000 <21.130 ADL5-D25 Zwitt AB206- Plad D ADL0010 2.700 97.000 <2 1.620ADL10-D1 AB206- Plad D ADL0012 6.400 90.000 <2 0.670 ADL12-D1 AB206-Plad D ADL0012 6.300 90.000 <2 0.850 ADL12-D2

TABLE 18 Exemplary anti-EPHA2 ADCs - PC3 cells SMLA Payload GI50 LD50Rmin GI50 LD50 Rmin Sample DAR (nM) (nM) (%) (nM) (nM) (%) AB206- 6.50.175 95.51 −68.678 16.622 177.783 −68.37 ADL1-D8 AB206- 6 <0.061 2.732−69.096 11.057 >400 −55.97 ADL1-D4 AB206-

3.046 >400 −49.287 3.877 >400 −79.835 ADL1-D14 AB206- 6.4 0.808 >400−64.516 13.291 >400 −71.987 ADL12-D1 AB206- 6.3 78.977 >400 17.3762.391 >400 −68.803 ADL12-D2 AB206- 4.6 71.73 >400 19.765 1.41 >400−29.33 ADL5-D19 AB206- 2.7 >400 >400 43.796 13.291 >400 −71.987 ADL10-D1AB206- 4.2 9.959 >400 2.441 3.877 >400 −79.835 ADL5-D15 AB206- 528.078 >400 20.041 >400 >400 77.524 ADL5-D25 AB206- 5.6 <0.061 >400−59.643 13.291 >400 −71.987 ADL1-D1 AB206- 6.3 1.003 >400 16.4652.391 >400 −68.803 ADL5-D2

indicates data missing or illegible when filed

Example 5

The in vitro and/or in vivo stability of exemplary anti-HER2 ADCs wasassessed as described below.

5.1 Total Antibody and Conjugated Payload of AB185-ADL1-D1 in Plasma

Following in vitro treatment (section 5.1.1) or in vivo treatment(section 5.1.2) with AB185-ADL1-D1, levels of total antibody (TAb) andconjugated payload (CP) were quantified in plasma by immunoprecipitationLC/MS/MS technique. Quantitation fit was linear with 1/x² weighting from0.5-100 μg/mL, and 0.5-500 ng/mL, in mouse plasma for TAb and CP,respectively. Plasma samples were mixed with stable-isotope labeleduniversal monoclonal human antibody internal standard (Slu™MAB, Sigma,St. Louis, Mo.) and 10 mM PBS, pH 7.4 and incubated at room temperaturefor one hour. Dynabeads® MyOne™ Streptavidin T1 magnetic beads(ThermoFisher Scientific) were added to the mixture and incubated for anadditional 30 min at room temperature. A magnetic stand was used to bindbeads, which were sequentially washed with CHAPS buffer and then PBSbuffer. Analytes of interest were eluted from the beads with 30 mM HCl.Separate eluent aliquots were used to quantify TAb or CP. For TAb,Tris-HCl, pH 8.3 was added, followed by trypsin and allowed to incubateovernight at 37° C. For the CP, 1M ammonium acetate was added, followedby deuterated payload internal standard, and cathepsin B, and allowed toincubate overnight at 25° C. Samples were analyzed by LC/MS/MS andquantified using area ratio of analyte to internal standard and comparedagainst respective calibration curve. To calculate CP concentration, theconcentration of TAb was corrected for molecular weights of payload,molecular weight of AB185-ADL1-D1 and DAR (drug-to-antibody ratio).

5.1.1 In Vitro Stability of AB185-ADL1-D1 in Mouse, Rat, and MonkeyPlasma

Pooled plasma (Bioreclamation) from mouse, rat, and monkey, and PBS werespiked with AB185-ADL1-D1 (4.81 mg/mL) to achieve a final concentrationof 50 μg/mL. Samples were incubated at 37° C., 95% humidity, 5% CO₂ for4 days in a manner to stabilize pH and minimize non-specific binding andevaporation. Aliquots were removed at 4, 24, 48, 72, and 96 hours andstored at −70° C. until processing. Samples were thawed and processedfor bioanalysis using methods described above (section 5.1) for totalantibody and conjugated payload. Time 0 was set to 100% for each matrix.The area ratio responses of the later time points were compared to time0 separately for each matrix. Percent remaining was calculated asfollows: (Area Ratio day X/Area Ratio time 0)*100=% remaining. The slopeof the line was calculated in Excel comparing the log of % remaining andtime point. Half-life was calculated in Excel by ln(2)/slope.

Total antibody from AB185-ADL1-D1 was found to be stable in buffer andin all species of plasma, exhibiting only modest precipitation over 4days (FIG. 12A). Conjugated (intact) payload D1 levels were maintainedover 4 days (FIG. 12B).

5.1.2 Snapshot Pharmacokinetics (PK) in Mouse Plasma Following Single IVDose of AB185-ADL1-D1

Mice (CB17-SCID) were treated with a single IV dose of AB185-ADL1-D1 at10 mg/kg or 20 mg/kg, followed by terminal collections (cardiacpuncture) in Lithium-Heparin tubes (n=3 per time point per dose group)at 24, 48, and 72 hours post dose. Blood was centrifuged for 5 min at5000 rpm at 4° C. to remove red blood cells and the plasma was stored at−80° C. until processing. Samples were thawed and processed forbioanalysis using methods described above (section 5.1) for totalantibody and conjugated payload.

Following a single IV dose of AB185-ADL1-D1, more than 400 ng/mL ofintact D1 payload remained conjugated to AB185 antibody at 72 hours postdose (FIG. 13). The AB185-ADL1-D1 ADC demonstrated improved stability ascompared to an alternate ADC carrying a payload with a similar mechanismof action. The thailanstatin A-based ADC reported in Puthenveetil et al.(Bioconjugate Chem. (2016) 27:1880-8) is relatively less stable, andshows complete bioconversion of payload by 72 hours (i.e., acetate iscompletely hydrolyzed).

5.2 Accelerated Stability Testing of AB185-ADL14-D1 and AB185-ADL5-D25

Freshly produced SMLAs (˜1 mg each in Eppendorf tubes, 4-5 mg/mL) werecentrifuged and measured by UV absorption (NanoDrop) at 280 nm todetermine the protein concentrations. Samples were then incubated in a37° C. waterbath and sampled at four time points, 0 (freshly prepared),1 day, 2 days, and 4 days. Samples taken at different time points werestored at −80° C. until the last sampling was completed. After thawingat 22° C., samples were analyzed with SEC, hydrophobic interactionchromatography (HIC), and reverse-phase-MS (RP-HPLC) for quantificationof antibody aggregation/fragments, and DAR. Percent aggregation,concentration, and DAR values (HIC and RP-HPLC) for AB185-ADL14-D1 andAB185-ADL5-D25 at four time points are shown in Table 19.

TABLE 19 Accelerated stability testing of select anti-HER2 ADCs Days @Aggregation Concentration DAR ADC Storage Buffer 37 C. (%) (mg/ml) HICRP-HPLC AB185- DPBS(pH 7.5) 0 0.68 4.99 4.3 4.32 ADL14-D1 1 1.18 4.984.29 4.31 2 1.31 5.12 4.33 4.32 4 2.08 5.02 4.28 4.27 AB185- DPBS(pH7.5) 0 0.84 5.05 4.54 4.22 ADL5-D25 1 1.14 4.16 4.44 4.19 2 1.41 4.524.74 4.19 4 2.43 3.98 4.64 4.12

Example 6

To determine whether cells treated with splicing modulator-based ADCs(i.e., the exemplary ADCs described herein) could produce neoantigenssuitable to prime naïve T-cells to transition to effector cells (i.e.,effector cells capable of targeting such neoantigens), the followingexperiments were performed.

6.1 RNA Sequencing and Protein Ligandome Experiment 6.1.1 Overview

Changes in the transcriptome were interrogated following ADC treatment.Two HER2-overexpressing cell lines, one engineered non-small-cell lungcancer line (NCI-H1568) and one HER2-amplified breast cancer line(HCC1954) were treated with ADC (AB185-ADL1-D1) at concentrationsintended to trigger robust aberrant mRNA splicing (4 nM and 1.3 nM,respectively). RNAseq experiments were performed at 24 hours posttreatment. These experiments demonstrated that multiple mRNA transcriptsexhibited altered splicing patterns.

To then interrogate whether the identified transcripts could betranslated and presented as neoantigens on tumor cell MHC1 complexes, atimepoint that would allow for translation of the mRNA transcripts, butthat would precede extensive cytotoxicity effects (as those may impaircollection of cell material for analysis) was selected. NCI-H1568 andHCC1954 cell lines were then treated with ADC at 3 nM for 48 hours andthe MHC1-bound peptidome was analyzed by LC-MS/MS. Peptidome data wasfiltered by primary peptide size to 8-14 amino acid fragments (definedas the size limit criteria for binding to MHC1) and then filteredagainst known human proteins. The identified peptides were characterizedas self antigens, regardless of under which conditions and in which cellline they were detected. All remaining peptide sequences were mappedagainst 3-frame translations of the RNAseq data. Using stringentcriteria for evaluating the peptide sequences (including exclusion of:any peptide identified in any untreated sample, any peptide that was notencoded in the canonical open reading frame, and any peptide that wasnot encoded by an exon-junction-spanning portion of the mRNA where theexon junctions were altered by treatment with AB185-ADL1-D1), fourMHC1-bound neopeptides were identified. For a schematic of an exemplaryRNA sequencing and protein ligandome experiment, see FIG. 14.

6.1.2 Detailed Methods 6.1.2.1 RNA Sequencing of ADC-Treated Cells

HCC1954 and HER2 overexpressing NCI-H1568 (ATCC) cells were plated at1×10⁵ cells per individual well of a 6-well tissue culture plate(Corning). Cells were treated with phosphate-buffered saline orAB185-ADL1-D1 at 1.3 nM and 4 nM, respectively, for 24 hours. Total RNAwas extracted from cells using an RNAeasy Mini kit (Qiagen), andassessed for quality and quantity (RNA 6000 Nano LabChip kit on a 2100Bioanalyzer, both from Agilent). Poly-A selected RNA-Seq libraries wereprepared according to standard Illumina protocols. cDNA libraries werechecked for quality and quantified using the DNA-1000 kit on a 2100Bioanalyzer (both from Agilent). Libraries were pooled and sequenced ona HiSeq2000 (Illumina) to obtain 101-base paired-end reads.

Raw sequence reads were aligned to the human reference sequence hg19 bySTAR 2.4.2a using two-pass alignment35, and isoform quantification wasperformed using Kallisto 0.42.4 (Bray et al. (2016) Nat Biotechnol.34(5):525-7) against GENCODE annotation v 25 mapped to GRCh37.Estimation of the retained intron count was performed by defining 6 nt(−3 nt from the splice site and +3 following the splice site)exon-intron boundary regions and counting alignments, which fullyoverlapped the regions. All raw junction counts, including those fromexon-intron boundaries, were pooled over all technical replicates percohort. Differential junction usage was assessed using a binomial z testfor differences in proportion between treated and untreated samplepools. Proportions were calculated on the basis of all splice junctions(and exon-intron boundaries) which shared a splice site, similar to thepercent spliced in (PSI) measurement. In order to preserve Gaussianassumptions, only those shared splice sites with total junction count(sum raw count over all junctions sharing that splice site) greater thanor equal to 10 reads in both treated and untreated cohorts wereconsidered for analysis. z-scores corresponding to a false discoveryrate (FDR)-corrected q value of ≤0.05 for treated (‘aberrant’) junctionsand ≤0.20 for untreated (‘canonical’) junctions were consideredsignificant.

Two or more splice junctions that share a splice site and have at leastone junction that is significantly upregulated in treated samples or atleast one junction significantly upregulated in untreated samples wereconsidered aberrant or canonical junctions, respectively. Both aberrantand canonical junctions were required to be present in an event for itto be considered differential splicing. In the case of intron-retentionevents, both exon-intron boundaries (5′ and 3′ splice sites) as well asthe junction bridging the two were required to be significant.

6.1.2.2 MHC1-Bound Peptidome of ADC-Treated Cells

HCC1954 and HER2 overexpressing NCI-H1568 (ATCC) cells were plated at1×10⁸ cells on 15 cm² tissue culture plates (Corning). Cells weretreated with phosphate-buffered saline or AB185-ADL1-D1 at 3 nM for 48hours. Cells were mechanically removed from the plates and processed asper Bassani-Sternberg et al. ((2015) Mol Cell Proteomics 14(3):658-73).Briefly, the procedure involved solubilization of the MHC-1 proteincomplexes from cellular membranes, enrichment of MHC-1 protein togetherwith its bound peptides by immunoprecipitation, selective elution andpurification of bound peptides, and identification and quantification ofpeptides by LC/MS/MS analysis. For immunoprecipitation the monoclonalantibody W6/32 purified from its hybridoma cell line HB-95TM was used.Mass spectrometric analyses were performed on a Q Exactive HFinstrument. Raw files acquired in this study were processed with theMaxQuant software suite (version 1.5.7.13) for peptide and proteinidentification and quantification using a human protein sequencedatabase (version 22017). In addition to this database, predictedtranscript variants based on RNAseq experiments of AB185-ADL1-D1-treatedHCC1954 and NCI-H1568 cells were included. MaxQuant performs 6-frametranslation and integrates obtained peptide sequences for matching withMS² spectra. Data were further manually filtered for peptide sequencesunique to AB185-ADL1-D1-treated samples as well as for peptides encodedby mRNA splice junctions modulated by AB185-ADL1-D1 treatment (FIG. 14).Four neopeptides were identified by a high-stringency filtering of theRNAseq and MHC-1-bound ligandome data. These were encoded by four genes.The protein sequences of the four neopeptides, referred to herein asNeoantigen 1, Neoantigen 2, Neoantigen 3, and Neoantigen 4, wereextended for the following experiments on triggering immune cellpriming. The extended protein sequence incorporates both the neopeptidesequence itself in addition to flanking amino acid sequences. Theextended protein sequence better facilitates the uptake of protein bydendritic cells and enables antigen presentation and T-cell priming inmodels with different HLA isotypes.

6.2 T-Cell Priming Experiment 6.2.1 Overview

This experiment was an in vitro reconstitution of the interactions thatoccur in normal human secondary lymphatic organs (e.g., tumor draininglymph nodes). In this experiment, monocytes were isolated fromperipheral blood mononuclear cells (PBMC) and were induced todifferentiate into dendritic cells (DC) through culturing in a cytokinecocktail. Following differentiation to mature DC, the mature DC werecultured with extended neopeptide sequences. During culturing, themature DC take up the peptides, process them into fragments, and presentthem on MHC1 for priming of CD8 T-cells. The DC were then mixed withadditional PBMC from the same donor and incubated for approximately 2weeks in a cocktail of cytokines to stimulate the activation of CD8T-cells. Following incubation, the cells were transferred to an ELISpotplate that had been re-coated with the peptides used for priming andre-stimulation of CD8 T-cells was monitored by secretion of IFNγ. TheELISpot plate was then processed and imaged, and the IFNγ spots werecounted to estimate the number of active CD8 T-cells. For a schematic ofan exemplary T-cell priming experiment, see FIG. 15.

6.2.2 Detailed Methods 6.2.2.1 Neoantigen-Mediated T-Cell Priming andActivation ELISpot

Bulk peripheral blood mononuclear cells (PBMC) were purchased frommultiple vendors (StemExpress, HemaCare, Precision Medicine) and wereused to generate antigen-reactive CD8+ T-cells as per Wölfl andGreenberg ((2014) Nat Prot. 9(4):950-66; FIG. 15). Briefly, frozen PBMC(5×10⁷ cells) were thawed and plated on T-75 tissue culture plastic(Corning) in RPMI1640, supplemented with 5% human AB serum and 1%penicillin/streptomycin for 2-3 hours. Non-adherent cells were removedand discarded, and adherent cells were cultured in RPMI1640/5% humanserum, supplemented with 20 ng/mL GM-CSF and 20 ng/mL IL-4 for 5 days.On day 6, media was further supplemented with 20 ng/mL TNF-α. On day 7,mature DC were collected, counted, and loaded with neopeptides that wereidentified from MHC1-peptidome experiments (FIG. 16). Cells wereseparated and loaded with each peptide at a final concentration of 10μg/10⁸ cells/mL and incubated for 1 day. On day 8, DCs were treated withmitomycin C at 20 μg/mL for 30 min and washed extensively. Concurrentwith the treatment of the DC, 10⁸ matched PBMC were thawed and washedwith RPMI1640. PBMC were then mixed with DC at a ratio of 10:1 in 10 mLRPMI1640/5% human AB serum, supplemented with IL-21 at 30 ng/mL in T25flasks. After 3 days of co-culture (day 11), an additional 10 mL offresh media was added and supplemented with IL-7 and IL-15 at a finalconcentration of 5 ng/mL. Following an additional 3 days of co-culture(day 14), an additional 10 mL of fresh media, supplemented with IL-7 andIL-15 at a final concentration of 5 ng/mL, was added and the co-culturewas moved to a T-75 flask. On day 17, an additional 20 mL of media,supplemented IL-7 and IL-15 at a final concentration of 10 ng/mL, wasadded and cells were transferred to a T-175 flask. On day 20, cells werecollected, and ELISpot assay was performed as per manufacturer'sinstructions (R&D Systems). Wells were pre-loaded using peptide at 10μg/mL final concentration per well, cell density at 2×10⁸ cells perwell, duplicate seeding, and overnight incubation. IFNγ spots weredeveloped and read on an AID ELISpot reader as per manufacturer'sinstructions (FIG. 17A-D).

Exemplary data from antigen priming experiments are shown in FIG. 17A-D.Donor-derived PBMC lots were differentiated into DC and primed withneopeptides as outlined in FIG. 15. Analysis of ELISpot data shows that,in healthy donor PBMC, priming of naïve T-cells by DC presentingneopeptides can result in antigen-specific expansion and maturation ofeffector (CD8) T-cell populations. These T-cell populations exhibitantigen-specific re-activation, as evidenced by IFNγ secretion only inthe presence of the antigenic peptide and not in the presence ofnon-priming peptides.

RT-qPCR

RNA was purified from cell lines using RNeasy Mini with DNaseI treatment(Qiagen) and 1-2 μg of RNA reverse transcribed using Superscript VILOreverse transcriptase (ThermoFisher Scientific) in 20 μL according tomanufacturer's instructions. RNA lysates were isolated and reversetranscribed using TaqMan Gene Expression Cells-to-CT Kit (ThermoFisherScientific) according to manufacturer's instructions. Quantitative PCRwas performed using TaqMan Gene Expression Master Mix (ThermoFisherScientific) with transcript probes targeting neoantigen junctionsduplexed with GAPDH RNA VIC-PL (ThermoFisher Scientific) and quantifiedusing the ΔΔCt method.

Example 7 7.1 RNA Sequencing and Protein Ligandome Experiment 7.1.1Methods

RNA sequencing and protein ligandome experiment was performed asdescribed in Example 6 (Section 6.1). For a schematic of an exemplaryRNA sequencing and MHC1 ligandome experiment, see FIG. 14.

7.1.2 Results

Twenty nine neopeptides were identified by a high-stringency filteringof the RNAseq and MHC1-bound ligandome data (Table 20). Four neopeptideswere selected and extended for the following experiments on triggeringimmune cell priming. The four selected neopeptides are shown in bold inTable 20.

TABLE 20 Neopeptides SEQ ID Junction Observed Neopeptide NO (HG19) GeneEvent type in  1 SPTLPPRSL 37 chr12: TUBA1C Intron retention H156849663470- 49663610:+  2 HPSIKRGLSSL 38 chr12: PPHLN1 Exon skipping H156842729776- 42781257:+  3 LLLPHHVL 39 chr12: TUBA1C Intron retention H156849663470- 49663610:+  4 RTAPGVRPPF 40 chr14: CFL2 Intron retention H156835182767- 35183743:-  5 RPQKSIQAL 41 chr10: WAC Intron retention H156828822963- 28823162:+  6 APAPPPLPA 42 chr17: GPS1 Intron retention H156880009840- 80011149:+  7 RPRPSFPVSL 43 chr7: EGFR Intron retention H156855087058- 55134942:+  8 RPKHGDGFSL 44 chr11: MED19 Intron retentionH1568 57472287- 57472444:-  9 GPAPGKTGL 45 chr7: HSBP1 Intron retentionH1568 75932393- 75933118:+ 10 EAARKGNSL 46 chr1: SCP2 Exon skippingH1568 53480715- 53504588:+ 11 RIKEKIEEL 47 chr9: SMC5 Exon skippingH1568 72897499- 72912881:+ 12 EIKKRFRQF 48 chr1: DNAJC8 Exon skippingH1568 28531860- 28541450:- 13 HESAAMAET 49 chr11: TMEM123 Exon skippingHCC1954 102272937- 102323254:- 14 ALKLKQVGV 50 chr1: CHTOP Exon skippingH1568 153610924- 153617539:+ 15 DLKKRHITF 51 chr13: MRPS31 Exon skippingH1568 41323417- 41331008:- 16 DVKRNDIAM 52 chr1: NFYC Exon skippingH1568 41213277- 41218822:+ 17 IPSDHILTPA 53 chr6: TAB2 Exon skippingH1568 149718900- 149720239:+ 18 TVFSTSSLK 54 chr11: SDHAF2 Exon skippingH1568 61197654- 61213412:+ 19 ITSCLLNF 55 chr5: HSPA9 Intron retentionH1568 137892555- 137893090:- 20 RASPVRGQL 56 chr7: MDH2 Intron retentionH1568 75677544- 75677893:+ 21 VVRKPVIAL 57 chr1: MRPS15 Exon skippingH1568 36923582- 36929406:- 22 LLSEKKKIS 58 chr6: VARS Intron retentionH1568 31750622- 31750872:- 23 APASKPRPRL 59 chr19: HMG20BIntron retention H1568 3573798- 3574380:+ 24 RYGQLSEKF 60 chr19: PDCD5Exon skipping HCC1954 33076813- 33078158:+ 25 VYISNVSKL 61 chr3: SELKExon skipping HCC1954 53920961- 53925796:- 26 LPTKETPSF 62 chr2: TMSB10Alt 3'ss HCC1954 85133241- 85133394:+ 27 GEAPPPPPA 63 chr17: CSNK1DIntron retention HCC1954 80223672- 80231181:- 28 LEEISKQEI 64 chr17:TAOK1 Exon skipping HCC1954 27804724- 27807385:+ 29 IYNHITVKI 65 chr4:ADD1 Exon skipping HCC1954 2886393- 2896308:+

The protein sequences of the twenty nine neopeptides were extended. Theextended protein sequence incorporates both the neopeptide sequenceitself in addition to flanking amino acid sequences. The extendedprotein sequence better facilitates the uptake of protein by dendriticcells and enables antigen presentation and T-cell priming in models withdifferent HLA isotypes. Amino acid sequences of the twenty nine extendedneopeptides are set forth in Table 21.

TABLE 21 Amino acid sequences of extended neopeptides SEQ Gene ID NOExtended neopeptide amino acid sequence* TUBA1C 66VDLEPTVIGELTSVTQVRSQGAGTGGLSWGGSAGHSPTLPPRSLSLLLLPHHVLQMKFALALTASSSTLSNSSQARKMLPITMPEGTTPLARRSLTSCWTEFASWLTSAPVFRASWFSTALVGELVLGSPRCSWNVSQLIMARSPSW SSPFTRRPRFPQL PPHLN167 APPRSHPSIKRGLSSL CFL2 68 MVRRARWPGGRGEARKAPRTAPGVRPPF WAC 69WVNCLFVSGRAAAGGGGGGAVPPYLELAGPPFLLLTLIRIGLGRRSGRAGGRAGTQCGGERGPGFAAFRPLRPFRRLRVCAVCVRGSALGRSVGLPRGGAAGAPFSSSPAPHPRRVLCRCLLFLFFSCHDRRGDSQPYQVPAEAGVEGLEGAGGGREGLLLERRPQKSIQALRCNTSETSTADPLKIPGLVPLALS SKV GPS1 70MPLPVQVFNLQVTSRGRPGPPRPRAPRHWGRAEVEQGRGACARSRSGTLRAGPPRAARVGGCRAEGASPPWLRAAIGGRRAAPAPPPLPAAHGRGSRP PRR EGFR 71QPAQPRTGAPARRPRPRPSFPVSLRSAAPPTGTAGGTGRFVLRPGESGAGGGGDAWDTGLQARRGTAAGTSGAPNRSQLSSLTFPAQLRRIGVSGRKPGAGGRLGPGSRTCAPRCLPRARRGPGAHPRGGRCPPAETALFREAEEGT QKYSLPSDPAGQAAF MED1972 FRLHTGPVSPVGGRRQMGRPKHGDGFSLQVCSFIMEQNG HSBP1 73GVVEITGEPPCSCRGEEEASRAGRAGGVRLKRGSRGPGELNVGPAPGKT GLLIPLLRNWECGSLLRALSALSCP2 74 KMGFPEAARKGNSL SMC5 75 LEARIKEKIEELQQALI DNAJC8 76EIKKRFRQFKQAVYKQ TMEM123 77 AHESAAMAETLQHVPS CHTOP 78 NRPSVQAALKLKQVGVMRPS31 79 KTDDLKKRHITFTLGCGIC NFYC 80 MKLDEDVKRNDIAMAI TAB2 81NSISQIPSDHILTPALFITFMTILDL SDHAF2 82 TVFSTSSLKLNQPQKYLKMKSWPC HSPA9 83AEEDRRKKVITSCLLNFNLSKAQS MDH2 84 RSFSTSAQVGQTRGGLQAEAPRPGPRASPVRGQLMRPS15 85 RGYVVRKPVIALSVKI VARS 86VDMDFGTGGQGAGPVGRGKDWSCTLAVHLLSEKKKISFSQIDRAWGGSQGTVLDKWGPGVVSELHPSAKEVSVGRNSVESLMTWAS HMG20B 87EKGSHEEEVRVPALSWGRPRAPAPASKPRPRLDLNCLWLRPQPIFLWKL RPRPVPAATPLTGPLPLPDCD5 88 RYGQLSEKFNRRKVMDS SELK 89 MVYISNVSKLCFSKM TMSB10 90NTLPTKETPSFLLNPHTSWVPRPHREAPRLRVGVAAPLQRPLPALHSH CSNK1D 91FGDIYLGEAPPPPPAARRPGPCGCQDQARSRKEVVAPAGSPRKSRHRRI VARTQRPLG TAOK1 92GSASDLLEEISKQEISF ADD1 93 QLIYNHITVKINLQGD * Underline indicates aminoacids derived from the canonical transcript open reading frame (i.e.,the canonical peptide sequence).

7.2 T-Cell Priming Experiment 7.2.1 Overview

For a schematic of an exemplary T-cell priming experiment, see FIG. 15.

7.2.2 Materials

Materials used for T-cell priming:

-   -   RPMI 1640 Medium (Thermo Fisher Scientific, Cat. No. A10491-01)    -   Penicillin/Streptomycin (100×, Thermo Fisher Scientific, Cat.        No. 15140122)    -   Human Serum Type AB (Sigma, Cat. No. H3667-100ML)    -   Recombinant Human IL-4 (Peprotech, Cat. No. 200-04)    -   Recombinant Human GM-CSF (Peprotech, Cat. No. 300-03)    -   Recombinant Human TNF-α (Peprotech, Cat. No. 300-01A)    -   Human IL-7 (Peprotech, Cat. No. 200-07)    -   Human IL-15 (Peprotech, Cat. No. 200-15)    -   Human IL-21 (Peprotech, Cat. No. 200-21)    -   Peptides (New England Peptide—95% purity, dissolved in DMSO to a        1000× stock at a concentration of 10 μg/μL)    -   ELISpot kit for Human IFN-γ (R&D Systems, Cat. No. EL285)

7.2.3 Methods 7.2.3.1 Generation and Maturation of HumanMonocyte-Derived Dendritic Cells and Loading of Neopeptides

Frozen or fresh PBMC were thawed and washed in pre-warmed RPMI mediaonce, and an aliquot of non-adherent cells was reserved for baseline TCRsequencing. PBMC were resuspended in 10 mL of dendritic cells (DC)medium (RPMI1640, supplemented with 5% human AB serum and 1%penicillin/streptomycin) at 5×10⁶ cells per mL, for a total of 50million cells. Cells were transferred to T-75 tissue culture plastic andincubated at 37° C., 5% CO₂ for 2-3 hours. After gentle shaking,non-adherent cells were removed and discarded, and adherent(monocyte-enriched) cells were washed three times with pre-warmed DCmedium. 10 mL of DC medium supplemented with a final concentration of 20ng/mL GM-CSF and 20 ng/mL IL-4 was added to adherent cells. Adherentcells were incubated at 37° C., 5% CO₂, at 100% humidity for 5 days. Onday 6, recombinant TNF-α was added to a final concentration of 20 ng/mLfor another 48 hours. On day 7, DC were collected and counted, thenloaded with neopeptides (each at 10 μg/1×10⁶ cells/mL) or no neopeptidecontrol. On day 8, cells were harvested as mature DC. Mature DC weretreated with mitomycin C at a final concentration of 20 μg/mL for 30 minat 37° C. followed by extensive washing before co-incubation withT-cells for priming.

7.2.3.2 Start of Co-Culture with Autologous T-Cells

On day 0, frozen or fresh were thawed and washed in pre-warmed mediaonce. PBMC were then resuspended and mixed with DC at a ratio of 10:1(10×10⁶:1×10⁶ cells) in 10 mL RPMI1640/5% human AB serum, supplementedwith IL-21 at 30 ng/mL in T25 flasks. After 3 days of co-culture (day3), an additional 10 mL of fresh media was added and supplemented withIL-7 and IL-15 at a final concentration of 5 ng/mL. Following anadditional 3 days of co-culture (day 6), an additional 10 mL of freshmedia, supplemented with IL-7 and IL-15 at a final concentration of 5ng/mL, was added and the co-culture was moved to a T-75 flask. On day 9,an additional 20 mL of media, supplemented IL-7 and IL-15 at a finalconcentration of 10 ng/mL, was added and cells were transferred to aT-175 flask. On days 11-12, cells were collected, ELISpot assay wasperformed as per manufacturer's instructions (R&D Systems), and TCRsequencing was performed (1×10⁶ cells). Wells were pre-loaded usingpeptide at 10 μg/mL final concentration per well, cell density at 2×10⁵cells per well, duplicate seeding, and overnight incubation. IFNγ spotswere developed and read on an AID ELISpot reader as per manufacturer'sinstructions.

7.2.4 Results

Exemplary data from antigen priming experiments are provided in Table22.

TABLE 22 T-cell priming data ELISpot SEQ ID HLA Donor response Gene NO(predicted binding) 1 +++ DNAJC8 76 A*31:01 / B*40:13 1 + TMEM 123 77A*02:01 2 +++ TMEM 123 77 A*02:01 2 + SCP2 74 B*39:06 6 + DNAJC8 76C*07:01 / B*27 7 +++ DNAJC8 76 0*07:02 / 0*06:02 8 +++ DNAJC8 76 0*07:018 + SMC5 75 C*07:01 / B*08:02 8 +++ SCP2 74 0*03:03

Donor-derived PBMC lots were differentiated into DC and primed withneopeptides as outlined in FIG. 15. Analysis of ELISpot data shows that,in healthy donor PBMC, priming of naïve T-cells by DC presentingneopeptides can result in antigen-specific expansion and maturation ofeffector (CD8) T-cell populations. These T-cell populations exhibitantigen-specific re-activation, as evidenced by IFNγ secretion only inthe presence of the antigenic peptide and not in the presence ofnon-priming peptides. The data also suggest that response to neopeptidescannot necessarily be predicted based on HLA.

Example 8 8.1 Identification of Neopeptides 8.1.1 Overview

To identify neopeptides resulting from splicing modulation, peptides 8to 11 amino acids in length and predicted to bind to MHC class I fromthe canonical and splicing-modulated proteome were matched separatelyagainst tandem mass spectra using a standard target-decoy approach.First, alternatively-spliced mRNA transcripts were translated andcompared against a reference proteome. Next, spectra that did not matchthe reference proteome were searched for matching novel peptides. For aschematic diagram of an exemplary process for identifying novel peptidesresulting from splicing modulation, see FIG. 14.

8.1.2 Methods 8.1.2.1 Creating A Database of Potential Neopeptides

To generate a database of potential neopeptides, junction-spanning orintron-containing peptides not found in a reference proteome wereidentified (see Li et al. (2011) Mol. Cell Proteomics10(5):M110.006536). Peptides derived from the reference proteome wereselected from manually reviewed protein sequences and downloaded. Inaddition, the proteins were filtered for junctions that are found innormal tissues. As an alternative approach, complete proteins may betranslated from splicing events, searched for junction-spanning orintron-containing peptides, and then matched by BLAST against thereference proteome.

To avoid unnecessary statistical tests, only peptides predicted aslikely to bind to MHC were used. Protein sequences (both from thereference proteome and from predicted translation of exon-skipped orintron-retained transcripts) were evaluated for high affinity binding toMHC1 alleles using NetMHCPan and/or MHCnuggets software. For bothsoftware packages, hits with strong or weak binding were chosen (i) byraw affinity binding prediction strength (500 nM threshold for weakbinding), or (ii) by selecting a large number of random real peptidesand identifying the distribution of predicted binding strengths for thepeptides. The predicted binding strength for any novel peptides werethen matched against this distribution. The thresholds for strong andweak binding measured by the latter method (method (ii)) were predictedbinding strengths within the top 0.5% or the top 2.0% of the random realpeptides, respectively (see Nielsen and Andreatta (2016) Genome Med.8(1):33). Currently, MHC prediction is restricted to peptide sequences 8to 11 amino acids in length and contained in the Immune Epitope Database(IEDB).

8.1.2.2 Identifying Splicing Modulation-Derived Transcripts by RNAseqand RiboSeq

Several neopeptides are derived from larger peptide domains (e.g.,TUBA1C), while peptide fragments from the remainder of the translatedregion may risk going undetected. This may be a consequence of eitherenhanced degradation of the undetected regions, MHC1 binding affinity,or technical challenges associated with detection by mass spectrometry(MS). Generally, RNAseq analysis will overestimate the number ofpredicted neopeptides (since not all alternatively-spliced transcriptswill be translated), while the MHC1-ligandome will underestimate thenumber of predicted neopeptides.

To establish a list of neopeptides, available splicingmodulation-derived variant splice junctions with bound ribosomes wereidentified, as a marker of translation to proteins (Andreev et al.(2017) Nucleic Acids Res. 45(2):513-26). RiboSeq or ribosomal profilingallowed for detection of mRNA sequences (and variant domains) that wereundergoing active translation, as evidenced by the presence of ribosomesbound to the transcript, as well as specific exon junction domains.

Example 9

9.1 In Vitro Analysis of Exemplary Anti-HER2 ADCs

9.1.1 Methods

HCC1954, NCI-N87, or MCF7 cells (ATCC) were plated in phenol-red freeRPMI+10% FBS (ATCC) or EMEM+10% FBS+insulin (0.01 mg/mL) (for MCF7) at2.5×10³ cells per well at 90 μL per well. Cells (n=3 wells percondition) were treated with conjugates in a 3-fold dilutiondose-response. After 24 hours, cells were lysed with 50 μL of CL buffer(IgePal CA-630, 5M NaCl, 1M Tris HCl 1M pH 7.4 in water) containing 25μL/mL of RNAsin (Promega) and incubated for 45 min at RT on a rocker.Resulting mixture (1 μL) was used to assess splicing modulation in aTaqman Fast Virus 1-Step MasterMix (Applied Biosystems) reversetranscription PCR reaction with the following Taqman primers accordingto the manufacturer's recommendations: SLC25A19 (Invitrogen,Hs00222265_m1); FBXW5 (IDT, forward primer: CACACCAGATCGGCATCAA (SEQ IDNO:37), reverse primer: CGATGATGTGTCCGTGTATGT (SEQ ID NO:38), probe:ATCCTGCCACACCAGATGACCAC (SEQ ID NO:39), dye: FAM, quencher: ZEN/IowaBlack FQ); TAOK1 (IDT, forward primer: CTGCTTCGGATTTACTAGAAGAGATA (SEQID NO:40), reverse primer: GCGTTCCCACAAAGGAATTG (SEQ ID NO:41), probe:TGCTGACTTTGGCTCTGCTTCCAT (SEQ ID NO:42), dye: FAM, quencher: ZEN/IowaBlack FQ); RPLPO (Invitrogen, Hs99999902_m1).

9.2 In Vivo Efficacy and Pharmacodyamics (PD) of Exemplary Anti-HER2ADCs 9.2.1 Methods—In Vivo Efficacy in NCI-N87 Xenograft Model

CB17-SCID mice (Charles River Laboratories) were kept in accordance withfederal, state, city, & AAALAC-accreditation policies on animal researchin the city of Cambridge, Mass. NCI-N87 human gastric carcinoma cellline was purchased from American Type Culture Collection (ATCC No.CRL-5822). Passage 14 cells were used for tumor experiments. Cells werelast tested for murine pathogens in September 2016 and Mycoplasma inDecember 2018.

Single cell suspension of 5 million cells per 0.1 mL (50%ATCC-modification-RPMI-1640-medium, Cat. No. A10491-01 (Thermo-FisherScientific); and 50% Corning Matrigel, Cat No. 356254 (VWR Scientific))was injected subcutaneously on the right lateral flank with a syringewith a 25 Ga needle. Mice were randomized on day 0 at a mean tumorvolume of 180-183 mm³, n=8 per group for efficacy and n=4 per group forpharmacodynamics (PD). On day 1, test articles were administered (10mg/kg) at a volume of 5 mL/kg intravenously every 7 days for 2 cyclesfor efficacy; a single course of treatment was used for PD assessment.Animals in the PD cohorts were euthanized at 24, 48, 72, and/or 96 hourspost-dosing of a single dose, depending on the test article. Tumorvolume and body weight were measured one or two times weekly until thetermination (day 61) of the study. Tumor volume was calculated using theformula length×width²/2. During the study, mice that reached endpoint(tumor size 20 mm³ in any direction or tumor volume starting bodyweight×100), or had a tumor ulceration or eye infection, were removed.

Compounds were formulated in biological safety cabinets. Test articlevehicle was phosphate buffered saline pH 7.4 (Cat No. A10010-049(Thermo-Fisher Scientific)). Groups were as set forth in Table 23.

Anti-tumor activity was calculated as follows:

% T/C formula=treated/control*100%

TGI formula=(vehicle average_(day X)−treated average_(day X))/vehicleaverage_(day X)*100,

wherein day X is day 22.

9.2.2 Methods—In Vivo PD (RT-qPCR)

RNA was purified from tumor samples using MagMAX-96 for Microarrays kitand MagMAX Express-96 Deep Well Magnetic Particle Processor(ThermoFisher Scientific). Tumor samples (size 50-100 mg) were collectedin RNAlater buffer and kept at −80° C. until RNA isolation. Tissuehomogenization was performed by adding 2-3 ceramic beads and 1 mL ofTri-reagent into a tube containing tumors, followed by disruption on anOmni Bead Ruptor 24 instrument (Omni International). RNA isolation wasdone according to the manufacturer's instructions.

RNA lysates were isolated and reverse transcribed using TaqMan FastVirus 1-Step Master Mixt (ThermoFisher Scientific) according to themanufacturer's instructions.

Quantitative PCR from cDNA was performed using TaqMan Gene ExpressionMaster Mix (ThermoFisher Scientific) with transcript probes targetingFBXW5 or TAOK1 junctions duplexed with RPLPO RNA VIC-PL (ThermoFisherScientific) and quantified using the ΔΔCt method.

TABLE 23 Groups Test Articles Test Articles (efficacy) (PD) Vehicle(PBS) Vehicle (PBS) TDM1 TDM1 trastuzumab trastuzumab (AB185) (AB185)AB185-ADL5-D2 AB185-ADL5-D2 AB185-ADL1-D4 AB185-ADL1-D4 AB185-ADL5-D15AB185-ADL5-D15 AB185-ADL14-D1 AB185-ADL14-D1

9.3 Results In Vitro Analysis:

Exemplary anti-HER2 ADCs were measured for antiproliferative activity intwo HER2-positive cell lines of different lineage (HCC1954 breast cancerand N87 gastric cancer) and a HER2-negative cell line (MCF7). Theefficiency of growth inhibition was measured (G150 potency), as well asthe depth of response (R min compared to control). Alternative splicingin a representative house keeping gene SLC25A19 was measured by qPCRafter treatment of the ADCs on the specified cell lines. G150 and depthof splicing response were measured. The results of the in vitro analysisare shown in Table 25.

In general, the tested anti-HER2 ADCs showed favorable potency on theantigen positive cell lines and minimal response for viability andsplicing in the antigen negative cell line. The ADCs differed in depthof response in cell killing and completeness of alternative junctionsplicing of SLC25A19. The ADCs fell into one of four categories on eachof the antigen positive cell lines: 1) high depth of response/lethalityand high splicing response; 2) high depth of response/lethality and lowsplicing response; 3) low depth of response/lethality and high splicingresponse; or 4) low cell lethality and low splicing response. SeveralADCs showed favorable, category 3 behavior on the N87 gastric cancercell line. AB185-ADL13-D4 was consistently lethal and showed strongdepth of splicing response in both HCC1954 and N87 cells. Thisconsistent antigen specific lethality is generally desirable for ADCsand the water soluble linker would allow for even higher drug loadingwith good conjugate specifications. Switching the linker to ADL23 (EVCtripeptide) surprisingly changed the profile in both cell lines.AB185-ADL1-D12 was also unique in that it is highly lethal on HCC1954,but the cellular potency of the payload is consistently low on a numberof solid and heme tumor lines. D12 is also the only payload in thisseries with a low efflux ratio. Thus, ADCs releasing this payload may beactive against multiple drug resistant cancers that express a targetantigen.

When splicing potency (1050 qPCR) is compared against cellular potency(G150 CTG) in HCC1954 breast cancer cells, the glucuronide linker andthe D12 payload showed favorable properties. The C6/07 carbamate switch(D8) payload and the dibasic payload (D5) showed cellular potency butdid not demonstrate the most efficient alternative splicing. Switchingfrom a dipeptide to a tripeptide linker with the D4 or the D1 payloadreduced alternative splicing efficiency yet cellular activity remainedhigh. See FIG. 18 (sized by cell lethality, shaded by depth ofalternative splicing response).

When splicing potency (1050 qPCR) is compared against cellular potency(G150 CTG) in N87 gastric cancer cells, the glucuronide linker (with D4)and the D12 payload showed favorable properties, as in HCC1954. Overall,all ADCs showed minimal splicing and potency on the HER2-negative cellline MCF7. Despite the relatively low cellular potency of the D12payload, the activity of anti-HER2 ADCs with this payload was high andthe payload showed a low efflux ratio. These properties couldpotentially translate to better efficacy in a multiple drug resistantcancer setting. See FIG. 19 (sized by cell lethality, shaded by depth ofalternative splicing response).

In vitro analysis was performed on additional anti-HER2 ADCs. Acomparison between anti-HER2 ADCs and corresponding payloads is shown inTable 26. When potency and lethality of anti-HER2 ADCs is comparedagainst stability and permeability of corresponding payloads (FIG. 20),AB185-ADL5-D15 demonstrated a favorable balance of potency, lethality,chemical stability, and catabolite permeability (e.g., for putativebystander killing). As described below, AB185-ADL5-D15 was also activein vivo and demonstrated potent anti-tumor activity in a NCI-N87xenograft model.

In Vivo Efficacy:

The human HER2-expressing NCI-N87 gastric cancer cell line and itsxenograft model was generated by inoculating NCI-N87 cells into SCIDmice to evaluate anti-tumor activity of exemplary anti-HER2 ADCs.Trastuzumab and TDM1 were used as comparators.

Anti-tumor effects of intravenous trastuzumab, TDM1, and four HER2 ADCsin NCI-N87 xenograft model of gastric carcinoma are shown in FIG. 21 andFIG. 23. Body weight effects are shown in FIG. 22 and FIG. 23. Testarticle or vehicle was given intravenously (IV) Q7D for 2 cycles. Datarepresent the mean±SEM (tumor volume, mm³) or ±SEM (body weight, %)(N=8). * p<0.0001 versus vehicle group on Day 22 using one-way ANOVAtest.

Tabular data for in vitro and in vivo efficacy in NCI-N87 is provided inTable 24. Tabular data represent percent (%) Tumor Growth Inhibition(TGI of Tumor Volume) or % T/C (Tumor Volume) (N=8). AB185-ADL5-D2 wassurprisingly active in vivo despite little lethality in vitro, anddemonstrated improved in vivo efficacy as compared to, e.g.,AB185-ADL14-D1.

TABLE 24 Efficacy of exemplary anti-HER2 ADCs in NCI-N87 Day 22 (invivo) NCI-N87 (in vitro) TGI (%) T/C (%) GI_(50 (nM)) LD_(50 (nM))R_(min) % Vehicle/ IV/ Q7Dx2 — — — — — TDM1/ 10 mg/kg/ 71 29 0.1 40 −37IV/ Q7Dx2 trastuzumab/ 4 96 >200 >200 98 10 mg/kg/ IV/ Q7Dx2AB185-ADL5-D2/ 73 27 0.2 >200 0 10 mg/kg/ IV/ Q7Dx2 AB185-ADL1-D4/ 68 320.1 >200 −22 10 mg/kg/ IV/ Q7Dx2 AB185-ADL5-D15/ 67 33 0.1 >200 −37 10mg/kg/ IV/ Q7Dx2 AB185-ADL14-D1/ −7 107 0.1 >200 −40 10 mg/kg/ IV/ Q7Dx2

Trastuzumab, an anti-human HER2 antibody and a standard of care therapyfor HER2-positive breast cancer, did not demonstrate anti-tumor activityin this model. However, TDM1, a second-line treatment containingtrastuzumab plus covalently linked DM1 (a cytotoxic agent) showedsignificantly better anti-tumor activity (TGI 71%) than trastuzumabalone (TGI 4%). Among the four ADCs tested, ABL185-ADL5-D2,AB185-ADL5-D15, and AB185-ADL1-D4 showed potent anti-tumor activity withTumor Growth Inhibition (TGI) of 73%, 67%, and 68%, respectively. Tumorsof all responding groups eventually started to grow again. No completeresponse was observed in any group, and one of eight animals in each ofthe following groups showed a partial response: TDM1, ABL185-ADL5-D2,and AB185-ADL5-D15. All reagents and test articles were well toleratedby mice and minimal body weight loss was observed. Overall, 3 out of the4 tested anti-HER2 ADCs demonstrated similarly potent anti-tumoractivity in comparison with TDM1 in the NCI-N87 tumor model withoutsignificant body weight loss.

In Vivo PD:

PD modulation of mRNA junctions by intravenous trastuzumab, TDM1, andfour anti-HER2 ADCs in NCI-N87 xenograft model of gastric carcinoma isshown in FIG. 24A-24D. RT-qPCR of FBXW5 (mature mRNA transcript) andTAOK1 (neojunction transcript) were monitored. Test article or vehiclewas given intravenously (IV) at 10 mg/kg. Animals (N=4 per group) werecollected at either 48 hours (FIG. 24A and FIG. 24B) or at the timesindicated (FIG. 24C and FIG. 24D). Tumors were isolated for RNAextraction and RT-qPCR. * p<0.05 versus vehicle using one-way ANOVA(FIG. 24A and FIG. 24B) or two-way ANOVA (FIG. 24C and FIG. 24D).

Neither trastuzumab nor TDM1 exhibited significant changes in theobserved splicing of either FBXW5 or TAOK1. AB185 conjugatesADL5-D2/ADL1-D4/ADL5-D15 all exhibited significant depletion of FBXW5,and AB185 conjugates ADL5-D2/ADL1-D4 exhibited significant increases inTAOK1. ADL5-D15 showed an increase in TAOK1, but did not reachstatistical significance. ADL14-D1 did not exhibit statisticallysignificant changes in abundance of either mRNA transcript.

TABLE 25 Characterization of exemplary anti-HER2 ADCs and correspondingpayloads Properties - HER2 ADCs antiproliferative activity antigen (+)cell #1 CTGIo CTGIo CTGIo Lethality/ GMean GMean Mean Splicing GI50 (nM)LD50 (nM) MinResp % Category HER2 SMLA HER2 SMLA HER2 SMLA ADC Batch(HCC1954, Percent Free screen screen screen Id Linker N87) DAR MonomerDrug Conc. HCC1954.1 HCC1954.1 HCC1954.1 AB185- mc-Val-Cit- 1, 3 6.2599% <1% 0.82 0.032 0.09 −78.471 ADL1-D12 PABC AB185- b-glucuronide 1, 14.47 99% <1% 0.78 0.059 0.275 −80.145 ADL13-D4 AB185- mc-Val-Cit- 1, 36.14 99% <1% 1.1 0.079 0.298 −81.189 ADL1-D5 PABC AB185- mc-Glu-Val-Cit-2, 3 5.84 99% <1% 1.03 0.175 1.526 −68.762 ADL23-D4 PABC AB185-mc-Val-Cit- 2, 3 6.75 99% <1% 1.13 0.117 0.741 −77.982 ADL1-D3 PABCAB185- mal-PEG2-Val-Cit- 1, 3 6.12 99% <1% 1.01 0.134 0.797 −75.134ADL22-D4 PABC AB185- mc-Ala-Ala-Asn- 2, 3 5.51 99% <1% 1.35 0.207 2.65−72.894 ADL21-D1 PABC AB185- mc-Ala-Ala-Asn- 4, 3 6.4 99% <1% 1.670.493 >100.000 −37.547 ADL21-D8 PABC AB185- mc-Ala-Ala-Asn- 1.55 99% <1%1.11 1.174 >100.000 14.652 ADL21-D4 PABC AB185- mal-CH2CH2—  3, ND 3.2598.80%   <1% 1.6 0.24 >200.000 −13.023 ADL15-D2 O—CH2CH2—(non-cleavable) Properties - HER2 ADCs antiproliferative activityantiproliferative activity splicing PD antigen (+) cell #2 antigen (−)cell antigen (+) cell #1 CTGIo CTGIo CTGIo CTGIo CTGIo CTGIo qPCR- GMeanGMean Mean GMean GMean Mean CellScreen GMean GI50 (nM) LD50 (nM) MinResp% GI50 (nM) LD50 (nM) MinResp % IC50 (nM) HER2 SMLA HER2 SMLA HER2 SMLAHER2 SMLA HER2 SMLA HER2 SMLA HER2 SMLA ADC Batch screen screen screenscreen screen screen screen SLC25A19 Id NCIN87 NCIN87 NCIN87 MCF7 MCF7MCF7 HCC1954.1 null AB185- <0.015 >100.000 −35.162 >100.000 >100.00053.606 0.144 ADL1-D12 AB185- 0.029 3.436 −55.322 >100.000 >100.00087.451 0.45 ADL13-D4 AB185- 0.04 >100.000 −32.663 >100.000 >100.00093.099 0.624 ADL1-D5 AB185- 0.049 >100.000 −49.316 >100.000 >100.00091.248 >100.000 ADL23-D4 AB185- 0.049 >100.000 −15.629 >100.000 >100.00091.495 >100.000 ADL1-D3 AB185- 0.057 >100.000 −25.808 >100.000 >100.00079.996 2.302 ADL22-D4 AB185- 0.072 >100.000 −46.965 >100.000 >100.00074.018 >100.000 ADL21-D1 AB185- 0.138 >100.000 −40.873 >100.000 >100.00094.772 >100.000 ADL21-D8 AB185- 0.235 >100.000 −36.848 >100.000 >100.00096.18 >100.000 ADL21-D4 AB185- 0.115 >100.000 −15.056 ADL15-D2Properties - HER2 ADCs splicing PD splicing PD splicing PD Properties -antigen (+) cell #1 antigen (+) cell #2 antigen (−) cell CorrespondingqPCR- qPCR- qPCR- qPCR- qPCR- Free CellScreen CellScreen CellScreenCellScreen CellScreen Payloads Mean GMean Mean GMean Mean target MinResp% IC50 (nM) MinResp % IC50 (nM) MinResp % binding HER2 SMLA HER2 SMLAHER2 SMLA HER2 SMLA HER2 SMLA SPA-ATS screen screen screen screen screenGMean SLC25A19 SLC25A19 SLC25A19 SLC25A19 SLC25A19 IC50 (nM) ADC BatchHCC1954.1 NCIN87 NCIN87 MCF7 MCF7 SF3B1 (WT) Id null null null null nullHELA.2 AB185- −85.744 0.095 −93.185 >100.000 −12.254 ADL1-D12 AB185-−72.455 0.177 −93.984 >100.000 −28.389 8.778 ADL13-D4 AB185- −65.7990.095 −91.243 >100.000 −10.455 ADL1-D5 AB185- −40.549 0.249−89.841 >100.000 −16.033 8.778 ADL23-D4 AB185- −38.621 0.199−78.218 >100.000 −24.342 ADL1-D3 AB185- −58.063 0.17 −91.266 >100.000−23.433 8.778 ADL22-D4 AB185- −37.396 0.201 −84.588 >100.000 −15.4636.085 ADL21-D1 AB185- −27.319 0.577 −70.144 >100.000 −12.798 5.921ADL21-D8 AB185- −16.088 5.097 −54.535 >100.000 −12.096 8.778 ADL21-D4AB185- 0.296 −83.148 ADL15-D2 Properties - Corresponding Free Payloadscellular potency cell cellular potency cell biochemical splicing assayline #1 line #2 qPCR-IVS- qPCR-IVS CTGIo-ATS CTGIo-ATS CTGIo-ATS ATSGMean ATS GMean GMean GMean GMean IC50 (nM) IC50 (nM) Ratio GI50 (nM)LD50 (nM) GI50 (nM) ADC Batch Ad2.1 Ad2.2 Ad2.1/ 72 h 72 h 72 h IdHELA.2 HELA.2 2.2 NCIH1650.1 NCIH1650.1 NCIH1568.1 AB185- 16.528 25.090.659 257.925 >10000.00 163.604 ADL1-D12 AB185- 20.887 17.321 1.20611.941 1960.398 6.369 ADL13-D4 AB185- 17.392 23.943 0.726 ADL1-D5 AB185-20.887 17.321 1.206 11.941 1960.398 6.369 ADL23-D4 AB185- 9.326 10.9280.853 ADL1-D3 AB185- 20.887 17.321 1.206 11.941 1960.398 6.369 ADL22-D4AB185- 17.568 15.853 1.108 12.121 >10000.00 7.363 ADL21-D1 AB185- 11.4039.034 1.262 19.15 >10000.00 6.568 ADL21-D8 AB185- 20.887 17.321 1.20611.941 1960.398 6.369 ADL21-D4 AB185- ADL15-D2 Properties -Corresponding Free Payloads cellular potency cell cellular potency cellline #2 line #3 cellular permeability CTGIo-ATS CTGIo-ATS CTGIo-ATSPerm. Perm. GMean GMean GMean Mean Caco- Mean Caco- LD50 (nM) GI50 (nM)LD50 (nM) 2 A-B Perm 2 B-A Perm ADC Batch 72 h 72 h 72 h (10e−6 (10e−6Id NCIH1568.1 THP1.1 THP1.1 cm/s) cm/s) ER AB185- 1193.037 202.487900.227 0.17 0.23 1.353 ADL1-D12 AB185- 45.485 10.943 33.329 0.1 1.7917.9 ADL13-D4 AB185- ADL1-D5 AB185- 45.485 10.943 33.329 0.1 1.79 17.9ADL23-D4 AB185- ADL1-D3 AB185- 45.485 10.943 33.329 0.1 1.79 17.9ADL22-D4 AB185- 65.737 11.085 41.408 0.14 2.05 14.64 ADL21-D1 AB185-68.125 8.745 38.969 0.128 2.027 15.88 ADL21-D8 AB185- 45.485 10.94333.329 0.1 1.79 17.9 ADL21-D4 AB185- ADL15-D2

TABLE 26 Exemplary anti-HER2 ADCs - HCC1954 cells Properties - HER2 ADCsantiproliferative activity antigen (+) cell #1 CTGIo CTGIo GMean Mean pH5.5 GI50 (nM) MinResp % Permeability Stability HER2 SMLA HER2 SMLACaco-2 A-B (t½, screen screen ADC Batch Id Linker DAR (10e−6 cm/s) 37C.) HCC1954.1 HCC1954.1 AB185- QVal-Ala 6.600 16 0.8 0.278 −47.381ADL5-D19 AB185- QVal-Ala 6.000 0.14 3.4 0.038 −46.265 ADL5-D25 AB185-QVal-Ala 4.140 0.6 4 0.206 −12.239 ADL5-D2 AB185- Val-Cit 2.800 0.1 4.50.050 −57.212 ADL1-04 AB185- QVal-Ala 4.020 1.8 5 0.254 −2.273 ADL5-D10AB185- Val-Cit 4.400 0.72 7 2.086 ADL1-D16 AB185- QVal-Ala 3.960 20 72.668 14.873 ADL5-D17 AB185- QVal-Ala 26 7 3.325 −38.285 ADL5-D34 AB185-QVal-Ala 4.000 3.4 7 0.912 −45.388 ADL5-D15

Example 10 10.1 Bioinformatics-Driven Indication/Target Selection

To identify target antigens highly expressed in low tumor mutationburden (TMB) tumors, as compared to normal tissue, a bioinformaticspredictive analysis was performed. This example explains 1) how eachfilter was defined; and 2) how to use the antigen/indication table toidentify suitable target/tumor pairs. For a schematic diagram of anexemplary target indication analysis, see FIG. 25.

10.1.1 Overview

Target antigens and indications were identified using a bioinformaticspredictive analysis. Data from TOGA was processed for tumor mutationburden (TMB), expression of target antigens for ADC delivery, and tumorimmune infiltration. Analyses, using published cutoffs of TMB andinfiltration to stratify predicted responders vs. non-responders toimmune checkpoint inhibitors (ICI), were performed to identifyindications and matching antigens. Surface antigen expression wasfurther filtered to assess a probabilistic population with antigenexpression at least 2-fold higher than any normal tissue (as evaluatedfrom the GTEx tissue expression database). Indications (or subsets of aTOGA lineage) with a suitable percentage of patients having one or moretarget antigens were further analyzed for mutation and immune metrics.Percentages of antigen positive patients having a tumor mutation burdenbelow an aggregate of 10 mutations/megabase and with a T-cellinfiltration score above 0.5 were assessed. Indications with an estimateof ˜50% of patients meeting these criteria are listed in Table 27.Patients in these cohorts (but not limited to these cohorts) may respondto treatment with a splicing modulator ADC (e.g., a splicing modulatorADC described herein).

10.1.2 Methods

Indication Prediction for Annals of Oncology Antigen Table Annotation:

Antigen: antigen gene name, only 59 genes likely to be good ADC targetsfrom Annals of Oncology literature review (see Moek et al. (2017) Annalsof Oncology, 28: 3083-3091) were included in the table (initial fullanalysis included all in silico surfaceome genes)

Cohort: TOGA cohort name

SUBTYPE: TOGA tumor subtype information

Cases_Total: The total number of cases

Cases.by.subtype: The number of cases by subtype

LTPM_median: Expression median value by Log 2(TPM+1)

LTPM_median_LowTMB: Expression median value by Log 2(TPM+1) for thecases with low TMB (<350)

LTMB_Median: Median value of Log 10(TMB)

LTMB_MAD: Robust measurement of standard deviation by MAD value of Log10(TMB)

Infil_Median: Median value of infiltration score according to PLoS OneT-cell signature

Infil_MAD: Robust measurement of standard deviation by MAD value ofinfiltration score according to PLoS One Tcell signature

Cor.with.Purity: Expression correlation with tumor purity

GTEX_Tissue: The GTEx tissue name with the highest median value ofexpression

GTEX_LTPM_Median: The median expression value of antigen in the GTExtissue with the highest median value of expression

Surfaceome.Filter*: Patient population after applying surfaceome filter:expression >2 fold of GTEx normal tissue with the highest median

LowTMB.Filter: Patient population after applying low TMB filter: <350per exome-seq (10 per mega bases2)

LowTMB.Surfaceome.Filter: Patient population after applying bothsurfaceome and tmb filters

Hilnfil.LowTMB.Surfaceome.Filter: Patient population after applyingthree filters: surfaceome expression filter, low tmb filter and highinfiltration filter

Surfaceome.Filter.Perc: Percentage of population after applyingsurfaceome filter: expression >2 fold of GTEx normal tissue with thehighest median

LowTMB.Filter.Perc: Percentage of population after applying low TMBfilter: <350

LowTMB.Surfaceome.Filter.Perc: Percentage of population after applyingboth surfaceome and tmb filters

Hilnfil.LowTMB.Surfaceome.Filter.Perc: Percentage of population afterapplying three filters: surfaceome expression filter, low tmb filter,and high infiltration filter

Probabilistic Estimation of Patient Population:

Resources Compiled:

-   -   1. Pan-can immune landscape paper (Ready et al. (2019) Journal        of Clinical Oncology, 37:992-1000)    -   2. TCGA RNA-seq/Exome-seq data bundle from Omicsoft    -   3. Immune gene signatures (Thorsson et al. (2018) Immunity, 48,        812-830)    -   4. GTEx data v7

Tumor mutation burden was calculated from the TCGA exome-seq mutationcalling using Omicsoft data. PLoS One T-cell signature (CD3D, CD3E, CD2)was used to quantify sample immune infiltration. Z score was generatedusing log 2(TPM+1) value from TCGA RNA-seq data.

Surfaceome.Filter: Probability of individual patient to have >2 foldexpression (log 2(TPM+1)) value comparing to the GTEx normal tissue withthe highest median was calculated as follows:

-   -   1. Identify the GTEx normal tissue that has the highest median        value for this gene among all GTEx normal tissues, assume it is        T_(h)    -   2. Synthesize GTEx data for this gene with 1000 patients for        each tissue using Synthetic Minority Over-sampling Technique        (SMOTE (Siemers et al. (2017) PLoS One, 12(7):e0179726)        maintaining the data distribution by tissue    -   3. Count the number of times (n times) this patient will be >2        fold of the GTEx synthesized data for the tissue T_(h), the        probability for this patient to be >2 fold of the GTEx normal is        n/1000    -   4. Calculate this probability for every patient    -   5. The sum of the individual probability is the probabilistic        estimation of the cohort population with >2 fold higher than the        GTEx normal    -   6. When applying other filters together, the sum of individual        probability will be only for the patients that pass the other        filters

10.1.3 Examples

Target antigens and indications identified using the exemplarybioinformatics predictive analysis described above are set forth inTable 27.

TABLE 27 Exemplary target antigens and indications Target AntigenIndication(s) MSLN ovarian cancer cervical cancer pancreatic cancer lungadenocarcinoma EPHA2 esophageal cancer FOLH1 prostate cancer CDH6 kidneycancer CEACAM5 colorectal cancer CFC1B pancreatic cancer ENPP3 kidneycancer FOLR1 ovarian cancer HAVCR1 kidney cancer esophageal cancer KITkidney cancer MET kidney cancer esophageal cancer MUC16 ovarian cancercervical cancer breast cancer SLC39A6 breast cancer prostate cancerSLC44A4 prostate cancer STEAP1 prostate cancer

1-908. (canceled)
 909. A compound of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein: R¹ is chosenfrom absent, hydrogen, C₁-C₆ alkyl groups, C₁-C₆ alkylalkoxy groups,C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylic acid groups, C₁-C₆alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzyl groups, C₃-C₈heterocyclyl groups, —O—C(═O)—(C₁-C₆ alkyl) groups, and —CD3; R² isabsent or a linker; R³ is chosen from hydrogen, C₁-C₆ alkyl groups,C₁-C₆ alkylalkoxy groups, C₁-C₆ alkylamino groups, C₁-C₆ alkylcarboxylicacid groups, C₁-C₆ alkylhydroxy groups, C₃-C₈ cycloalkyl groups, benzylgroups, C₃-C₈ heterocyclyl groups, and —O—C(═O)—(C₁-C₆ alkyl) groups;R⁴, R⁵, and R⁸ are each independently chosen from hydrogen, hydroxylgroups, —O—(C₁-C₆ alkyl) groups, —O—C(═O)—(C₁-C₆ alkyl) groups, andC₁-C₆ alkyl groups; R⁶ and R⁷ are each independently chosen fromhydrogen, —O—R¹⁷, —O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₆ alkyl groups,—NR¹⁵R¹⁶, and a linker; R¹⁵ and R¹⁶ are each independently chosen fromhydrogen, R¹⁷, —C(═O)—R¹⁷, and —C(═O)—O—R¹⁷; R¹⁷ is chosen fromhydrogen, C₁-C₆ alkyl groups, C₃-C₈ cycloalkyl groups, benzyl groups,and C₃-C₈ heterocyclyl groups; and Z″ is chosen from

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, R¹⁶, and R¹⁷ are eachindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups,—NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆alkylalkoxy groups, benzyl groups, and C₃-C₈ heterocyclyl groups;wherein at least one of R⁶ and R⁷ is hydrogen; wherein if R² is alinker, then neither R⁶ or R⁷ is a linker, and if R⁶ or R⁷ is a linker,then R² is absent; and wherein if Z″ is

and R² and R³ are H, then R¹ is neither H nor methyl.
 910. The compoundof claim 909 or a pharmaceutically acceptable salt thereof, wherein: R¹is chosen from absent, hydrogen, C₁-C₄ alkyl groups, C₁-C₄alkylcarboxylic acid groups, C₁-C₄ alkylhydroxy groups, and C₃-C₈cycloalkyl groups; R² is absent or a linker; R³ is chosen from hydrogen,C₁-C₄ alkyl groups, C₁-C₄ alkylalkoxy groups, C₁-C₄ alkylcarboxylic acidgroups, and C₁-C₄ alkylhydroxy groups; R⁴ is chosen from hydrogen,hydroxyl groups, —O—(C₁-C₄ alkyl) groups, —O—C(═O)—(C₁-C₄ alkyl) groups,and C₁-C₄ alkyl groups; R⁵ is chosen from hydrogen, hydroxyl groups,—O—(C₁-C₄ alkyl) groups, and C₁-C₄ alkyl groups; R⁶ is chosen fromhydrogen, —O—R¹⁷, —O—C(═O)—R¹⁷, C₁-C₄ alkyl groups, and a linker; R⁷ ischosen from hydrogen, —O—R¹⁷, —O—C(═O)—R¹⁷, —O—C(═O)—NR¹⁵R¹⁶, C₁-C₆alkyl groups, —NR¹⁵R¹⁶, and a linker; R⁸ is chosen from hydrogen,hydroxyl groups, —O—(C₁-C₄ alkyl) groups, and C₁-C₄ alkyl groups; R¹⁵and R¹⁶ are each independently chosen from hydrogen, R¹⁷, —C(═O)—R¹⁷,and —C(═O)—O—R¹⁷; R¹⁷ is chosen from hydrogen, C₁-C₆ alkyl groups, C₃-C₈cycloalkyl groups, benzyl groups, and C₃-C₈ heterocyclyl groups; Z″ ischosen from

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R¹⁵, R¹⁶, and R¹⁷ are eachindependently substituted with 0 to 3 groups independently chosen fromhalogens, hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups,—NR¹⁵R¹⁶, C₃-C₈ cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆alkylalkoxy groups, benzyl groups, and C₃-C₈ heterocyclyl groups;wherein at least one of R⁶ and R⁷ is hydrogen; wherein if R² is alinker, then neither R⁶ or R⁷ is a linker, and if R⁶ or R⁷ is a linker,then R² is absent and wherein if Z″ is

and R² and R³ are H, then R¹ is neither H nor methyl.
 911. The compoundof claim 909 or a pharmaceutically acceptable salt thereof, wherein: R¹is chosen from absent, hydrogen, methyl, and C₁-C₄ alkylcarboxylic acidgroups; R² is absent or a linker; R³ is chosen from hydrogen and C₁-C₄alkylcarboxylic acid groups; R⁴ is chosen from hydrogen, hydroxylgroups, —O—(C₁-C₄ alkyl) groups, —O—C(═O)—(C₁-C₄ alkyl) groups, andC₁-C₄ alkyl groups; R⁵ is chosen from hydrogen and hydroxyl groups; R⁶is hydrogen; R⁷ is hydrogen; R⁸ is chosen from hydrogen and hydroxylgroups; and Z″ is chosen from

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ are each independentlysubstituted with 0 to 3 groups independently chosen from halogens,hydroxyl groups, C₁-C₆ alkyl groups, —O—(C₁-C₆ alkyl) groups, C₃-C₈cycloalkyl groups, C₁-C₆ alkylhydroxy groups, C₁-C₆ alkylalkoxy groups,benzyl groups, and C₃-C₈ heterocyclyl groups; and wherein if R² and R³are H, then R¹ is neither H nor methyl.
 912. The compound of claim 909,wherein the compound is chosen from a compound of formula

and pharmaceutically acceptable salts thereof.
 913. The compound ofclaim 909, wherein the compound is chosen from a compound of formula

and pharmaceutically acceptable salts thereof.
 914. The compound ofclaim 909, wherein the compound is chosen from a compound of formula

and pharmaceutically acceptable salts thereof.
 915. The compound ofclaim 909, wherein the compound is chosen from a compound of formula

and pharmaceutically acceptable salts thereof.
 916. The compound ofclaim 909, wherein the compound is chosen from a compound of formula

and pharmaceutically acceptable salts thereof.
 917. The compound ofclaim 909, wherein the compound is chosen from a compound of formula

and pharmaceutically acceptable salts thereof.
 918. A pharmaceuticalcomposition comprising the compound of claim 909, or a pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable carrier. 919.A method of treating a subject having or suspected of having aneoplastic disorder, comprising administering to the subject atherapeutically effective amount of the compound of claim
 909. 920. Themethod of claim 919, wherein the neoplastic disorder is a leukemia, alymphoma, or a myeloma.
 921. The method of claim 920, wherein themyeloma is multiple myeloma.
 922. A method of reducing or inhibitinggrowth of a tumor in a subject having or suspected of having aneoplastic disorder, comprising administering to the subject atherapeutically effective amount of the compound of claim
 909. 923. An-L-D compound, wherein D is chosen from compounds of claim 909 andpharmaceutically acceptable salts thereof; and L is chosen from linkerscomprising a cleavable amino acid unit that is covalently attached to Ddirectly or through an optional additional spacer unit.
 924. The -L-Dcompound of claim 923, wherein D is chosen from compounds of claim 2 andpharmaceutically acceptable salts thereof.
 925. The -L-D compound ofclaim 923, wherein D is chosen from compounds of claim 3 andpharmaceutically acceptable salts thereof.
 926. The -L-D compound ofclaim 923, wherein the cleavable amino acid unit comprises valine (Val)attached to citrulline (Cit), wherein Cit is covalently attached to Ddirectly or through an optional additional spacer unit.